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COF»«IGHT DEPOSm 



AN INTRODUCTION TO 

BACTERIAL DISEASES 
OF PLANTS 



BY 

ERWIN F. SMITH 

In Charge of Laboratory of' Plant Pathology, 
Bureau of Plant Industry, 
United States Department of Agriculture, Washington. D. C. 

^lember of the National Academy of Sciences and of the American Academy of 
Arts and Sciences; Fellow of the American Philosophical Society; Ex-President: 
Society for Plant Morphology and Physiology (1901), Society of American 
Bacteriologists (1906), Botanical Society of America (1910). American Phyto- 
pathological Society (191 7), etc. 



Ecrire I'hlstoire d'uiie science nouveUe, c'cst se voiwr 
a d'eteniels recomweticcmciifs—Madaine Duclaiix 



PHILADELPHIA AXD LONDON 

W. B. SAUNDERS COMPANY 

1920 




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Copyright, 1920, by W. B. Saunders Company 



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PRINTED IN AMERICA 

KOV -9 lS20 

©CU604016 



To the memory of 

Charles F. Wheeler and Volney M. Spalding: 

modest American men of science, strong idealists and splendid 
teachers. 

Wheeler was born in New York; studied in Mexico Academy and the University 
of Michigan; served in the Civil War; was 20 years in a Michigan country drug- 
store, which he made a center of fine intelligence; was instructor in botany in the 
Michigan Agricultural College for 12 years; and, finally, for 8 years research worker 
in the United States Department of Agriculture. 

Spalding was born in New York; pursued high school and academic studies in 
Ann Arbor, Michigan; was teacher of botany in the University of Michigan for 28 
years, broken only by studies in Germany under Detmer, Pfeffer and Brefeld; was 
investigator for 6 years in the Carnegie Desert Ijaboratory at Tugson, Arizona; 
and, finally, endured a long period of forced inaction in Southern California, 
retaining, however, his clear mind and his scientific interests to the end. 

Wheeler studied critically the flora of a State, Spalding changed the type of 
botanical teaching in our secondary schools. Each was my friend for nearly 40 
years. The first showed me how to study flowering plants, opened my eyes to 
the wonders of wood and field and was my companion in a thousand delightful 
rambles. From him I had also my first lessons in French. The second taught 
me how to stud}' the parasitic fungus and where to find its literature, often read- 
ing it with me when it was in foreign tongues. Each was devoted to the micro- 
scope and to the laboratory method. Each served his generation faithfully and 
now sleeps with his fathers. 

They reached a multitude of students whose gratitude remains, and so, in the 
lovely words of Simonides, they lie 

ayrjpauTO} xP^I^^^ol evXoyiri 



PREFACE 



The manuscript of this book was completed for publi- 
cation in 1915 and, in general, that year may be taken as the 
date of the outlook, but here and there, where it seemed most 
necessary, it has been revised down to the end of 1919. 

Those who seek for completeness in these pages will not 
find it. The book is in no senes a monograph, but only, as its 
title indicates, an introduction to the subject. 

While the book has been made primarily for laboratory 
use under the guidance of a competent teacher, who will add to 
it or subtract from it as he desires, it is believed that many 
persons not students may find in it various things of interest, 
and partly with this wider public in view it has been illustrated 
very fully. 

This book is the result of 35 years of reading and 25 years 
of diligent laboratory and field investigation. More than 
most books, it is the product of experiment. There is scarcely 
a line or a statement in it that has not recjuired more than one 
experiment. It is also largely the product of a single labora- 
tory, that is to say, 8 of the 14 organisms here selected for 
special study were named by the writer (one with a colleague), 
two were worked out by others in his laboratory (Bacillus 
carotovorus and Bacterium maculicolwm) , and of the remaining 
four, all of Appel's statements have been verified with addi- 
tions under Bacillus phyiophihorus, many additions have been 
made to Pammel's statements under Bacterium campestre, 
the entire body of description has been worked up for Bacterium 
mori, and some additions have been made to Bacillus amylovorus. 
Moreover, all but 35 of the 650 illustrations are from this 
laboratory. 

A majority of the photographs in the book were made 
by James F. Brewer and the remainder by the writer. Fre- 
quently we worked together. In case of particularly good 



VI PREFACE 

photographs involving special technic I have sometimes men- 
tioned the maker and the method used, since every student 
should learn to make his own photographs and photomicro- 
graphs if he wishes to excel. Nearly all the photomicrographs 
were made on the little upright stand (Fig. 55) because I wished 
to demonstrate to the student that excellent results can be ob- 
tained with very simple apparatus if one is only willing to take 
pains. 

The book was written at the earnest request of teachers 
and by their judgment it will stand or fall. It is the first 
treatise of its kind in the world and, therefore, I trust, that 
evidences of crudity will be excused. Not being a teacher, I 
have been in doubt many times how best to present the difficult 
subject. 

I shall be glad to receive criticisms and suggestions looking 
toward a second edition and also notes, photographs, cultures, 
fresh and dry specimens and separates of papers from all parts 
of the world, that I may be able to continue my monographic 
studies. 

Erwin F. Smith. 

Washington, D. C, 
August, 1920. 



CONTENTS 



PART I 

A CONSPECTUS OF BACTERIAL DISEASES 
OF PLANTS 

Page 

Distribution Among Flowering Plants 4 

Period of Greatest Susceptibility 8 

What Governs Infection 12 

How Infection Occurs 15 

Time Between Infection and Appearance OF the Disease 16 

Recovery from Disease 17 

Agents of Transmission 19 

Extra-vegetal Habitat of the Parasites 34 

Morphology and Cultural Characters of the Parasites 35 

Action of the Parasite on the Plant 41 

Reaction of the Plant — Color-changes, Blights, Decays, Distortions, 

Overgrowths 48 

Prevalence and Geographical Distribution 51 

Methods of Control 68 



PART II 
METHODS OF RESEARCH 

Reference Books 76 

Apparatus 77 

For Preparation of Culture Media 77 

For Isolation and Care of Cultures 80 

For Preparation and Study of Sections 81 

For the Hot House and Inoculation Experiments 86 

For the Photographic Room 89 

Uses of Culture Media 99 

Preparation of Culture-Media 100 

Technic of Isolation 107 

Care of Cultures 109 

Study of Cultures 110 

Methods of Inoculation Ill 

Time and Place of Inoculation 112 

Care of Inoculated and Control Plants 113 

Preparation of Sections 114 



Vlll CONTENTS 

Page 

Staining Methods 116 

Care of Specimens 119 

Preparation of Illustrations 120 

Photographs 120 

Photomiciographs 123 

Lumiere Plates 126 

Planars 126 

Drawings 126 

Paintings 129 

Card Catalogies and Systems of Filing 130 



PART III 
SYNOPSIS OF SELECTED DISEASES 

I. The Cucurbit Wilt 132 

Type 132 

Cause — Bacillus tracheiphilus EFS 132 

Technic 135 

Determine 137 

For the Organism 137 

Morphology 137 

Cultural characters 138 

Response to Non-nutritional Environment 139 

For the Disease 139 

Signs 139 

Histology 139 

Variability 141 

Transmisi-ion 141 

Literature 145 

II. Black Rot of Crucifers 145 

Type 145 

Cause—Bacterium campestre (Pammel) EFS 147 

Technic 149 

Determine 154 

For the Organism 154 

Morphology 154 

Cultural Characters 155 

Response to Non-nutritional Environment 155 

For the Disease 156 

Signs 156 

Histology 156 

Variability 157 

Transmission 159 

Means of Prevention 159 

Literature 159 

III. Stewart's Disease of Maize 160 

Type 160 



CONTENTS IX 

Page 

Cause — Aplanobader s^fu'arfi (EFS.) McCulloch 161 

Technic 163 

Determine 167 

For the organism 167 

Morphology 167 

Cultural Characters 168 

Re&ponse to Non-nutiitional Enviionment 168 

For the Disease 169 

Signs 169 

Histology 170 

Variability 172 

Transmission 174 

Means of Prevention 176 

Literature 176 

IV. The Brown Rot of Solanaceae 177 

Type 177 

Cause — Bacterium aolanacenrum EFS 182 

Technic 188 

Determine 189 

For the Organism 189 

Morphology 189 

Cultural Chaiacters 193 

Response to Non-nutritional Environment 195 

For the Disease 195 

Signs 195 

Histology 196 

Variability 198 

Transmission 199 

Literature 200 

V. Bacterial Canker of Tomato 202 

Type 202 

Cause — Aplanobader luichiganense EFS 205 

Technic 207 

Deteimine 212 

For the Organism 212 

Moiphology 212 

Cultural Chaiacteis 213 

Response to Non-nutritional Environment 214 

For the Disease 215 

Signs 215 

Histology 215 

Variability 215 

Transmission 216 

Host Plants 219 

Literature 222 

VL Jones' Soft Rot of Carrot, Etc 223 

Type 223 

Cause — Bacillus carotoi'orus L. R. Jones 230 

Technic 241 



X CONTENTS 

Page 

Determine 243 

For the organism 243 

Morphology 243 

Cultural Characters 246 

Response to Non-nutritional Environment 249 

For the Disease 251 

Signs 251 

Histology 251 

Vai lability 251 

Transmission • 252 

Literature 252 

Vll. Bacterial Black Rot of the Potato 253 

Type 253 

Cause — Bacillus phytophthorus Otto Appel 257 

Technic 263 

Determine 268 

For the Organism 268 

Moiphology 268 

Cultural Chaiacteis 269 

Response to Non-nutrituonal Environment 272 

For the Disease 274 

Signs 274 

Histology 274 

Variability 277 

Transmission 278 

Literature 278 

Vm. The Bean Blight 280 

Type 280 

Cause — Bacterium phaseoli EFS 285 

Technic 287 

Determine 291 

For the Organism 291 

Morphology 291 

Cultural Characters 291 

Response to Non-nutritional Environment 293 

For the Disease 294 

Signs 294 

Histology 294 

Variability 295 

Transmission 295 

Literature 299 

IX. McCulloch's Cauliflower Spot 300 

Type 300 

Cause — Bacterium maculicoluni Lucia McCuUoch 304 

Technic 310 

Determine 311 

For the Organism 311 

Morphology 311 

Cultural Characters 312 



CONTENTS XI 

Page 

Response to Non-nutritional Environment 312 

For the Disease 312 

Signs 312 

Histology 313 

Variability 313 

Transmission 313 

Literature 313 

X. The Angular Leaf Spot OF Cotton 314 

Type 314 

Cause — Bacterium malvacearum EFS i . 321 

Technic 330 

Determine 333 

For the Organism 333 

Morphology 333 

Cultural Characters 333 

Response to Non-nutritional Environment 335 

For the Disease 335 

Signs 335 

Histology 336 

Variability 336 

Transmission 337 

Literature 339 

XL The Mulberry Blight 340 

Type 340 

Cause — Bacterium mori Boyer and Lambeit emend. EFS 342 

Technic 351 

Determine 354 

For the Organism 354 

Moiphology 354 

Cultural Characterfe 354 

Respoase to Non-nutritional Environment 355 

For the Disease 356 

Signs : 356 

Histology 357 

Variability 358 

Transmission 358 

Literature 358 

XII. Fire-blight of Apple, Pear, Quince, Etc 359 

Type 359 

Cause — Bacillus amylovorus (T. J. Burrili) Trevisan 367 

Technic 374 

Determine 380 

For the Organism 380 

Morphology 380 

Cultural Characters 380 

Response to Non-nutritional Environment 380 

For the Disease 380 

Signs 380 

Histology 381 



Xll CONTENTS 

Page 

Vaiiability 383 

Transmission 384 

Eradication of the Disease 385 

Literature 387 

XIII. The Olive Tubercle 389 

Type 389 

Cause — Bacterium savastanoi EFS 391 

Technic 404 

Determine 408 

For the Organism 406 

Morphology 406 

Cultuial Characters 407 

Response to Non-nutiitional Environment 409 

For the Disease 409 

Signs 409 

Histology 409 

Variability 410 

Transmission 410 

Literature 411 

XIV. The Crown Gall 413 

Type 413 

Cause — Bacterium tumefaciens Smith and Townsend 421 

Technic 432 

Determine 451 

For the Oiganism 451 

Morphology 451 

Cultural Characters 451 

Response to Non-nutiitional Environment 457 

For the Disease 459 

Signs 459 

Histology 460 

Variability 468 

Transmission 469 

Literature 471 

PART IV 
MISCELLANEOUS 

I. Notes on Some ADDnioNAL Diseases 473 

II. Suggestion op Subjects for Special Study : , . . . 474 

III. Production of Tumors in the Absence op Parasites 477 

IV. Speculations on the Chemical and Physical Stimuli Underlying 

TUMOR-FORMATION 510 

V. On The Production of Teratosis in the Absence of Tumors and 

OF Parasites 574 



CONTENTS Xlll 

PART V 
GENERAL OBSERVATIONS 

Page 

On Subsidiary Studies 633 

On Seeing Things 635 

On Experimentation 637 

On Beginning Work Thoughtlessly 638 

On Interpretation of Phenomena 640 

On Repetition of Experiments — Other People's, One's Own 640 

On Publication 643 

On Clearness in Presentation 643 

On Completeness of Presentation 645 

On Brevity of Statement — When Brevity is not Desirable 646 

On the Ethics of Research 648 

On Keeping One's Own Counsel 654 

On Team Work 656 

On Sharing Credits 657 

On Attending Meetings and Keeping Up Membership in Societies, and 

ON BEING Generally Public-Spirited and Helpful in Science .... 659 

On Rest and Rbcre.a.tion 660 

Index 663 



LIST OF ILLUSTRATIONS 



Frontispiece : Prof. Thomas J. Burrill discoverer of the-first bacterial disease of 

plants (pear blight). Photograph in 1884 (at 45), handwriting in 

1915 (at 76). 

Page 

1. Savastano. Cavara, Wakker, Arthur, and Waite. Photographs of early 
workers on bacterial diseases of plants. All except of Savastano are of 

the period when they made their first investigations 2 

2. Mango fruit-cluster attacked by Doidge's bacterial spot disease 3 

3. Bacterial leaf-spot of mango from South Africa. M nat. size 9 

4. Coconut bud-rot of the West Indies 9 

5. Cross-section of a banana fruit-stalk attacked by Rorer's disease 11 

6. Angular leaf-spot of cucumber due to Baderiuni tachnjmans Smith 

and Brj-an 14 

7. Cucumber stem showing white film and cracks due to Bacterium 

lachrymans 14 

8. Gelatin stab culture of Bacterium lachrytnat^s 15 

9. Small gelatin colony of Bacterium lachrymans for marginal appearance. . 15 
10, 11. Recovery of tomatoes from an attack of Bacterium solanacearum EFS 18 

12. Head of wheat showing bacterial black chaff disease (Kansas, 1915).. . . 19 

13. Agar poured plate colony of black chaff bacterium {Bacterium trans- 

lucens var. undulosum Smith, Jones and Reddy). No. 273, Nebraska. 20 

14. Stalk- and glume-striping in black chaff of wheat. No. 268, Kansas. . . 22 

15. Bacterial exudate from glumes in black chaff of wheat. A natural 

infection on Montana spring wheat 23 

16. Black chaff' of wheat, showing a pure culture glume-infection done with 

No. 20 24 

17. Black chaff of wheat, showing ordinary ap|>earance of the yellow colonies 

on agar plates. No. 20, McKinney, Texas 26 

18. Black chaff of wheat, showing internal markings of surface colonies on 

agar. No. 662 from Monticello, Illinois 27 

19. Same as fig. 18 but No. 678 from El Reno, Oklahoma 28 

20. Black chaff of wheat: gelatin colonies of Bad. translucens var. undulosum, 

showing dry liquefaction pit.' No. 252 from Republic, Missouri 29 

21. Black chaff" of wheat, showing thin, secondary margin of two gelatin 

colonies. Medium magnification 31 

22. A, B. Black chaff of wheat. Same series as fig. 21 but older and 

more highly magnified 32 

23. Blade of a cucumber leaf attacked by angular leaf-spot showing tear- 

drop ooze of Bacterium lachrymans Smith and Bryan 37 

24. Gelatin colonies of Bacterium lachrymans 38 

25. Colonj^ markings of Bacterium lachrymans on: (A) gelatin and (B) agar. 39 

XV 



XVI LIST OF ILLUSTRATIONS 

Page 

26. A. Crystals formed by Bacterium lachrymans. B. Ditto by Bacterium 

solanacearum 40 

27. Ardisia leaf showing swollen serratures occupied by bacteria 41 

28. Bacterial cavity in leaf -tooth of Ardisia 41 

29. Like figure 28, but showing the water-pore 42 

30. A. Leaf of Pavetta angustifolia from Java showing knots. B. Same 

without knots 42 

31. Cross section of fig. 30A in vicinity of a nodule 43 

32. Bacterial cavities in leaf-knot on Pavetta angustifolia 43 

33. Detail from another nodule in same series as figure 32 44 

34. Bacterial masses from nodule on leaf of Pavetta angustifolia 45 

35. A. O'Gara's disease in head of western wheat grass. B. The same 

showing a knee-shaped bending of the culm. C. D. Hutchinson's 

East Indian (Punjab) wheat disease 47 

36. Shoots developing from the middle of a healthy tomato leaf 50 

37. Schizomycete of Japanese basket-willow disease — Agar plate colony 

showing internal structure when viewed by oblique light 53 

38. Kernels of wheat (No. 271A, Kansas, 1917) attacked and shriveled 

by the black chaff bacterium 56 

39. Head of wheat from Kansas (No. 478) showing the basal glume rot 

due to Bacterium, atrofaciens. McCulloch 57 

40. Glumes and kernels of wheat attacked by the basal glume rot 58 

41. Citrus canker on grape fruit leaves [due to Bacterium citri (Hasse) Jehlej. 

Fully developed 59 

42. Citrus canker on grape fruit leaf. Early stage 60 

43. Citrus canker on stems 61 

44. Section through a young bacterial canker on a grape fruit leaf 62 

45. Costa Rican pseudo-canker of citrus 63 

46. Costa Rican pseudo-canker of citrus. A detail from figure 45 64 

47. Scab on Florida citrus leaf due to Cladosporium citri 65 

48. Bacterial citrus canker enlarged to show the translucent border surround- 

ing old leaf-scabs 66 

49. Celery rot, due to Bacillus apiovorus Wormald 67 

50. A. Kernels of wheat developing Bacterium translucens var. unduloswm 

on nutrient agar. B. The same freed from the black chaff bacteria by 

formaldehyd 70 

51. Braun's new seed-wheat treatment, less harmful to the grain than 

ordinary treatments 72 

52. Electric centrifuge 79 

53. Freezing microtome 82 

54. Electric warm-plate 83 

55. Zeiss photomicrographic stand and small upright camera 85 

56. The Rutter kettle and other apparatus of the sterilizing chamber 87 

57. Simple home-made device for steaming infected soil 88 

58. The Crandall Model View Camera, Z}-i by A}'i inches 91 

59. Camera stand recommended by the writer 93 

60. Horizontal view of figure 59 93 

61. Diagram of photographic room and dark room used by the writer 96 

62. Cucumber plant wilted by Bacillus tracheiphilus EFS 133 



LIST OF ILLUSTRATIONS XVll 

Page 
£3. Squash plant wilted by Bacillus tracheiphilus 133 

64. Muskmelon plant wilted by Bacillus tracheiphilus 133 

65. Bacterially infected cucumber leaf gnawed by beetles. The infection 

preceded the gnawing 134 

66. Sound leaf locally infected by the gnawing of infected insects. The 

infection followed the gnawing 134 

67. Plant infected throughout as a result of insect gnawings like those 

shown in figure 66 135 

68. Flagellate rods of Bacillus tracheiphilus: a, b, X2000; c, XlOOO 136 

69. Enlarged colony of Bacillus tracheiphilus viewed by oblique transmitted 

light 136 

70. Like fig. 69 but less magnification: internal colony markings of Bacillus 

tracheiphilus on an agar-poured plate, visible by oblique transmitted 
light. Smooth on surface and homogeneous by direct transmitted 
light 138 

71. Agar buried and surface colonies of Bacillus tracheiphilus by reflected 

light ". 140 

72. Leaf stalk of an infected squash in cross-section 141 

73. Infected cucumber stem in cross-section 142 

74. A, Inner bundle of figure 73 enlarged; B, outer bundle of figure 73 

enlarged 142 

75. Infected cucumber stem in longitudinal section. Bundle destroyed. . . . 143 

76. Infected cucumber stem in longitudinal section showing empty and 

occluded spiral vessels 143 

77. Bacillus tracheiphilus highly magnified. From an infected cucumber 

vessel 143 

78. Cross-section of segment of a bacterially invaded pitted vessel. From a 

cucumber 144 

79. Field of cabbage in Wisconsin destroyed by black rot due to Bacteritun 

campestre (Pammel) EFS 145 

80. Early stages of water-pore infection in cabbage. Natural size 146 

81. Vertical section through an infected water-pore on cabbage 146 

82. Section parallel to surface of a cabbage leaf-tooth showing occluded and 

empty water-pore 147 

83. Upper stoma (water-pore) of the preceding enlarged, showing the rod- 

shaped (bacterial) mass blocking the stoma 147 

84. Inoculated cabbage leaf showing black-veined marginal spots due to 

water-pore infection 148 

85. Middle of a cabbage leaf showing black-vein disease due to stem in- 

oculation 148 

86. A, B. Cabbage leaf-stalks in cross-section showing black bundles, due to 

Bacterium campestre 149 

87. Black bundles in fleshy part of kohlrabi, due to Bacterium campestre. . . . 149 

88. A, B. Flagellate rods of Bacterium campestre 150 

89. A, B. Colonies of Bacterium campestre on agar. XlO 150 

90. Buried and surface colonies of Bacterium campestre by direct transmitted 

light 151 

91. Agar colonies of Bacterium campestre by oblique transmitted light 152 

92. Cultures of Bacterium campestre and Bacterium phaseoli in Dunham's 

solution 153 



XVlll LIST OF ILLUSTRATIONS 

Page 

93. Tyrosin crystals in milk fermented by Bacterium campestre 153 

94. Cross-section of a small cavity in a cauliflower stem, due to Bacterium 

campestre 154 

95. Cauliflower bundle in longitudinal section showing disorganization due 

to Bacterium campestre 154 

96. Turnip root in cross-section showing a bundle attacked by Bacterium 

campestre 155 

97. Beginning of a bacterial cavity in a turnip root 156 

98. Single cell of turnip root showing Bacterium campestre, occupying the 

intercellular spaces 158 

99. Longitudinal section showing a vessel and cells of a turnip root occupied 

by Bacterium campestre 158 

100. Large sweet-corn plant destroyed in the field by Aplanohacler stewarti 

(EPS) McC 160 

101. Flint field-corn, showing white top due to Aplanobacter stewarti 162 

102. Aplanobacter stewarti oozing from cut bundles (cross-section) of a sweet- 

corn stem : 163 

103. Aplanobacter stewarti oozing into water from vascular bundle of a 

sweet-corn stem — longitudinal section 163 

104. Corn plant from Chula Vista, California, showing dwarfing, white top 

and suckering due to Aplanobacter stewarti 164 

105. Yellow spots on inner husk of sweet corn due to Aplanobacter stewarti. . 164 

106. Cross-section of corn-husk from an inoculated plant showing bacterial 

masses in the tissues 165 

107. Stewart's disease of sweet corn, a detail of figure 106 166 

108. Infected small bundle at extreme base of a corn kernel. Cross-section.. 167 

109. Cross-section of a larger bundle at the same level as figure 108 168 

110. Longitudinal section showing infected vessels, etc., in periphery of a 

corn kernel, at the level of the radicle 169 

111. Infected vessels in periphery of sweet-corn kernel at the level of figure 

110. It shows Aplanobacter stewarti forming a small cavity 169 

112. Inoculated young sweet-corn plant attacked by Aplanobacter stewarti. . . 170 

113. Sweet-corn plant inoculated when young, and diseased when old 171 

114. A, B. Two types of surface colony in Aplanobacter stewarti 172 

115. Agar plate showing buried colonies of Aplanobacter stewarti and one 

coming to the surface. Also crystals 173 

116. Longitudinal section of two infected corn stems showing brown node 

and yellow striping 174 

117. Appearance under the microscoj>e of cross-section of empty and infected 

(stained) bundles in a maize stem 174 

118. Single disorganized bundle of an infected maize stem, enlarged. Bac- 

terial mass stained deep red 175 

119. Potato attacked by Bacterium solanacearum EFS. From a field near 

Washington, D. C 177 

120. Early Rose potato inoculated with Bacterium solanacearum. Late 

stage 178 

121. Inoculated young tomato. Early stage. Leaves of inoculated shoot 

reflexed 179 

122. A later stage of figure 121. showing incipient roots on inoculated stem . . 179 



LIST OF ILLUSTRATIONS XIX 

Page 

123. Tobacco leaf attacked by Bacterium solaiiaceaniin 180 

124. Inoculated young tobacco plant attacked by Bacterium solanacearum. . . 181 

125. Dwarf nasturtium attacked by Bacterium solanacearum, 182 

126. A. Common garden balsam attacked by Bacterium solanacearum. B. 

Stem of same enlarged 183 

127. Dwarfing of Ricinus due to Bacterium solanacearum 184 

128. A, B. Sunflowers destroyed by Bacterium solaiiacearum 185 

129. Cross-section of a mature potato tuber showing vascular invasion by 

Bacterium solanacearum 186 

130. Petiole of Tropaeolum majus (nasturtium) attacked by Bacteriutn 

solanacearum. Inoculation on stem at X 187 

131. Dwarfing of Helianthus annuus due to Bacterium, solanacearum. Checks 

at right and left 189 

132. Appearance of Bacterium solanacearum on agar poured plates 190 

133. Fluid character of colonies of Bacterium solanacearum on agar plates. . . . 191 

134. Flagellate rods of Bacterium solanacearum: a, East Indian origin; b, 

American origin 192 

135. Stabs of Bacterium solanacearum in nutrient + 10 gelatin 192 

136. Cross-section of a young potato tuber showing removal of starch from 

the areas infected by Bacterium solanacearum 193 

137. Infected potato stem in longitudinal section. Early stage. Bacterium 

solanacearum confined to a single vessel 194 

138. Beginning of a bacterial cavity (around a vessel) in stem of a potato 

plant inoculated on a leaflet with Bacterium solanacearum 196 

139. Empty and bacterially occluded vessel in a potato plant. Result of a 

stem inoculation 197 

140. Bacteria from same series as figure 139, highly magnified 198 

141. Tomato stem in cross-section, showing origin and structure of two 

incipient roots — result of an inoculation 199 

142. Tyloses in vessels of a potato stem attacked by Bacterium solanacearum. 

Bacteria at X 200 

143. Tomato plant attacked by Aplanobacter michiganense EFS, as result of 

needle-prick inoculation 20-3 

144. Tomato plants inoculated one month with a pure culture of Aplanobacter 

michiganense. Massachusetts organism. 1915, colony A. Check 
plants in background 204 

145. Stems of tomato plants showing only a slight tendency to form roots 

when inoculated (over 3 months) with Aplanobacter michiganense . . . 205 

146. Tomato leaf showing irregular withering of leaflets due to Aplanobacter 

michiganense. Inoculated in the stem 206 

147. Tomato stem in cross-section showing an incipient root destroj-ed from 

within (the black part) by Aplanobacter michiganense 207 

148. A, B. Tomato stem in cross-section showing large cavities in the phloem 

as a result of inoculating Aplanobacter michiganense 208 

149. Longitudinal section of a tomato stem attacked by Aplanobacter michi- 

ganense in the sieve-tube region 209 

150. Cross-section of a small group of sieve-tubes in a tomato stem showing 

sieve-plates, and disintegration of the phloem by Aplanobacter michi- 
ganense 209 



XX LIST OF ILLUSTRATIONS 

Page 
15 L 1, 2. Green tomato fruits attacked by Aplanobacter michiganense. From 

a hothouse in Massachusetts in 1915 210 

152. Same series as figure 150, showing loss of green color and swelling of 

stems before rupture. (See figure 153 for later stage.) 211 

153. Crack on a tomato stem due to Aplanobacter michiganense which was 

inoculated farther down 211 

154. Green tomato fruit oozing Aplanobacter michiganense as result of a stem 

inoculation 212 

155. Tomato plant infected with Aplanobacter michiganense through broken 

roots 212 

156. Stomatal leaf-infection due to Aplanobacter michiganense 213 

157. Longitudinal section of a tomato leaf showing bundle disorganization 

due to Aplanobacter michiganense 214 

158. Detail of a leaf-bundle infection in a tomato sprayed with Aplanobacter 

michiganense 215 

159. Rods of Aplanobacter michiganense highly magnified 216 

160. Aplanobacter michiganense induced to grow in acid agar by presence of 

another organism 217 

161. The new Massachusetts potato disease (net-necrosis) by reflected 

light 218 

162. Cross-section of stem-end and eye-end of a diseased potato tuber (net- 

necrosis) from Massachusetts 220 

163. Thin section of a third tuber photographed by transmitted light 221 

164. Raw carrot destroyed by Bacillus carotovorus L. R. Jones. Check-half 

sound 224 

165. Like right half of figure 164, but after it was accidentally dropped 226 

166. Separation of cells of carrot due to Bacillus carotovorus 225 

167. A. Bacillus carotovorus streaked on flabby vs. turgid carrot — 3d day; 

B. Ditto, 6th day. In each case one check is omitted 227 

168. Raw potato tuber attacked by Bacillus carotovorus 228 

169. Potato shoot attacked by Bacillus carotovorus 228 

170. Green cucumber rotted by BaaZ^ws caro^ownis. Check-half sound 229 

171. Calla lily rot due (?) to Bacillus carotovorus 230 

172. Enlarged cross-section of figure 171 at base 231 

173. A detail of figure 172 highly magnified, showing bacterial disorganiza- 

tion of the cell-wall 232 

174. Cork layer formed by a potato tuber under a rot spot due to Bacillus 

carotovorus 233 

175. Cross-section of a potato stem attacked by Bacillus carotovorus 233 

176. Flagellate rods of Bacillus carotovorus 234 

177. Agar-poured plate colonies of Bacillus carotovorus (?). Stock culture 3a 

(long in my laboratory) 235 

178. Do. Jones' original stock of Bacillus carotovorus. 3a (received from 

Madison, Wisconsin, in 1920.) 236 

179. Gas from Bacillus carotovorus in potato juice 237 

180. Bacillus carotovorus on gelatin — magnified surface colony 24 hours old, 

showing fimbriate margin 239 

181. Bacillus carotovorus on gelatin — fimbriate margin of a surface colony 

3 days old — liquefied at left 239 



LIST OF ILLUSTRATIONS XXI 

Page 

182. Bacillus caroloi'orus in gelatin — buried colonies 24 hours old, showing 

root-like outgrowths 240 

183. A, Bacillus carotovorus and B, Bacillus apioporus in gelatin stabs 241 

184. Behavior of soft rot bacteria in peptone beef bouillon with ten per 

cent ethyl alcohol 242 

185. Rot of celery due to Bacillus apiovorus Wormald 244 

186. Resistance of potato shoots to Bacillus apiovorus 245 

187. A, B, C. Three photoniicrograi)hs of flagella of Bacillus aroidece Towns- 

end 247 

188. Bacillus aroideae after 8 days on raw carrot 248 

189. A. B. Gas in milk produced by Bacillus aroideae — all CO- 250 

190. Curling of potato leaflets when stem is attacked by Bacillus phyla ph- 

thorus Appel 253 

191. Potato stems attacked by basal stem rot due to Bacillus phytophthorus — 

48 hours 254 

192. Same as figure 191 but after another 48 hours 254 

193. Inoculated potato plant destroyed by Bacillus phytophthorus — 7 days. . . 255 

194. Potato shoot attacked by Bacillus phytophthorus — 43 hours 256 

195. Shoots of White McCormick potato inoculated with Bacillus phytoph- 

thorus in 1915. Control plants in the background 258 

196. Same as figure 195, but two days later 259 

197. AVoody base of figure 193 two days later. Sound mother tuber at 

right 260 

198. Bacillus phytophthorus: same as figure 195, but the inoculations are on 

older, more resistant shoots 261 

199. Same as in figure 195, but 19 days later — new shoots growing up from 

the undestroyed base 262 

200. Enlarged cross-section of figure 193, well above the inoculated part. 

Bacteria in a vessel 263 

201. Bacillus phytophthorus attacking the cut surface of a raw potato 264 

202. Lenticel infection in a potato tuber due to Bacillus melanogenes P. and M. 265 

203. Verv early stage of decay in potato tuber under a sound skin — lenticel 

infection like figure 202 266 

204. Flagellate rods of Bacillus phytophthorus 267 

205. a. Gelatin colonies of Bacillus carotovorus with ib) those of Bacillus 

phytophthorus for comparison. Natural size 269 

206. Gelatin plate cultures of Bacillus phytophthorus from a South Carolina 

potato. 1917 270 

207. Bacillus phytophthorus on gelatin — a surface colony magnified to show the 

fimbriate margin. 271 

208. Bacillus phytophthorus in gelatin — 2 buried colonies, magnified 271 

209. Bacillus phytophthorus. — Small buried colony in gelatin 5 days at 16° C, 

showing lenticulate coronal colonies in the gelatin 272 

210. Effect of ethyl alcohol on Bacillus phytophthorus and Bacillus carotovorus 

in peptone bouillon 273 

211. Inoculations showing virulence of Bacillus phytophthorus (Appel I) 

after 13 years on culture media 275 

212. Gelatin plate culture of Bacillus carotovorus. B. Cross-section of 

Tropaeolum stem attacked by Bacterium solanacearum 276 



XXll LIST OF ILLUSTRATIONS 

Page 

213. Portion of an immature bean leaflet showing an early stage of infection 

with Bacterium phaseoli EFS 280 

214. Pure-culture spray inoculation of Bacterium phaseoli on an immature 

bean leaflet 281 

215. Bean leaflet attacked by Bacterium, phaseoli. From a garden in Wash- 

ington, 1908 282 

216. Spots on- a bean leaflet due to a pure culture stomatal infection of 1914. 

Chlorophyll persisting around the spots 283 

217. Distortions of bean leaves due to infection of the young veins by Bac- 

terium phaseoli 284 

218. Portion of a bean pod enlarged to show earliest visible stage of stomatal 

infection. 8th day 285 

219. Pure culture pod inoculation of bean blight {Bacterium pluiseoli). A 

single spot enlarged 286 

220. Bean pod sprayed 14 days with Bacterium phaseoli and showing spots. 

1914. Natural size 287 

221. Base of bean pod attacked by Bacterium phaseoli. From Idaho, 1914. . 288 

222. Same series as figure 218, but some days later 288 

223. Cross-section of a sprayed bean leaf prior to collapse — a stomatal 

infection 289 

224. A portion of figure 223 more highly magnified — two stomata visible. . . . 290 

225. Cross-section of a sprayed bean pod (stomatal infection) showing epi- 

dermis pushed up by the bacteria 291 

226. Cavities in outer tissues of a bean pod due to Bacterium phaseoli. Needle- 

prick infection of 1897 292 

227. Flagellate rods of Bacterium phaseoli 294 

228. Effect of sunlight on Bacterium phaseoli 295 

229. Buried and surface agar colonies of Bacterium, phaseoli 296 

230. Agar poured plate colonies of Bacterium phaseoli showing internal 

markings by direct transmitted light — surface smooth 298 

230*. Agar poured plate of Bad. phaseoli showing ringed colonies 299 

231. Cauliflower leaves spotted by Bacterium maculicolum McCulIoch 301 

232. Cauliflower heads attacked by Bacterium maculicolum 302 

233. Agar poured plate of Bacterium maculicolum from a Sanford, Florida, 

cauliflower 303 

234. Cross-section of an inoculated infected cauliflower head 305 

235. Leaf spot of New York cauliflower 307 

236. Surface and buried colonies of Bacterium maculicolum from the New 

York cauliflower (figure 235) 309 

237. Flagellate rods of Bacterium maculicolum 313 

238. A natural infection of Bacterium malvacearum EFS on cotton leaves. 

From South Carolina, 1903 314 

239. Bacterium malvacearum on cotton — a natural infection of leaves and 

bracts. From Arizona, 1914 315 

240. Inoculated cotton leaves. Stomatal infections. Veins diseased. Bac- 

teria smeared on. Early stage. 1915 316 

241. Results obtained in 1915 by inoculating cotton leaves in various ways 

with pure cultures of Bacterium malvacearum - . 318 

242. Inoculated cotton leaves. Stomatal infection due to Bacterium mal- 

vacearum. Time, 6 weeks. Bacterial suspension sprayed on. 1915. 319 



LIST OF ILLUSTRATIONS XXlll 

Page 

243. Cross-section (under a stoma) of an angular leaf spot of cotton in an 

early stage of infection, i.e., before collapse and shrinking of the 
spot 320 

244. Cotton leaf inoculated from a "windowed" colony of Bacterium mal- 

vacearum. Time, 53 days 321 

245. Green cotton bolls accidentally attacked by Bncterium malraceariim. 

Hothouse. 1904 322 

246. Middle stage of bacterial boll spot of cotton due to Bacterium mal- 

vacearum — lint involved a little 323 

247. Cotton stems attacked by Bacterium malcacearum — "black arm" of 

cotton 324 

248. Flagellate rods of Bacterium malvacearum , 325 

249. Agar-poured plates showing development of Bacterium malvacearum 

five days after exposure of one-half to bright sunlight for 2 minutes. 
Contrast with figure 228 326 

250. Effect of freezing on Bacterium malracearum 328 

251. Young (3-day) agar-plate colonies from one of the spots shown in 

figure 240. Plating of March 20, enlarged 329 

252. Agar colonies of Bacterium malvacearum to show fugitive mottling. 

Plating of March 22. Photographed March 25 330 

253. Photograph of "windowed" colonies (Nos. 4 and 5 of March 24) as 

they appeared on March 26 when filled in. See figure 256 332 

254. Third day agar plate of Bacterium, malvacearum, showing "windowed" 

and feebly mottled surface colonies 332 

255. Accidental inoculations of Bacterium malvacearum on cotton bolls. 

Early stage. 1915 333 

256. Mottled colonies of Bacterium malvacearum on agar plate (for later 

appearance see figure 253). Also three buried colonies 334 

257. Spatulate, finger-like down-growths of Bacterium malvacearum in soft- 

ened gelatine 335 

258. Streak cultures of Bacterium, malvacearum and Bacterium ])haseoli on 

Loffler's solidified blood serum 336 

259. -Milk culture of Bacterium malvacearum 337 

260. Tyrosin crystals from old litmus milk culture of Bacterium malvacearum 338 

261. Section of diseased cotton leaf extruding bacteria through a stoma in the 

upper epidermis. From the field 338 

262. American mulberry shoot inoculated with Bacterium mori Boyer and 

Lambert emend. EFS. Leaves spotted and distorted 341 

263. Distortion of South African mulberry leaves due to Bacterium mori 342 

264. Inoculated shoots of mulberry showing sunken stripes and extrusion of 

bacteria in cirri from lenticels 343 

265. South African mulberry twigs killed by Bacterium mori 344 

266. French Morus nigra attacked by Bacterium mori 345 

267. French Morus alba attacked by Bacterium, mori 346 

268. Cross-section of stem-tissues of mulberry showing inner bark destroyed 

by Bacterium mori as the result of a natural infection. Arkansas, 
1908 347 

269. a, Same as figure 268 but enlarged; h, cavity in a stem resulting from 

an inoculation , 349 



XXIV LIST OF ILLUSTRATIONS 

Page 

270. Leaves and stems of mulberry from Georgia attacked by Bacterium 

mori 350 

271. South African mulbeny disease due to Bacterium mori. Section of a 

leaf showing a bacterial pocket 352 

272. A. Flagella of the South African mulbeny parasite. B. The South 

African mulberry organism in the tissues 353 

273. Flagellate rods of Bacterium, mori from the United States 355 

274. a, b. Appearance of agar poured plate colonies of Bacterium mori with 

different lightings. X 10 356 

275. A single surface colony of Bacterimu mori further enlarged by oblique 

light to show internal markings 357 

276. Healthy and blighted branch of a Maryland pear tree. A detail 360 

277. Branch of an apple tree, showing flowers, fruits and shoots blighted by 

Bacillus amylovorus (Burrill) Trevisan 361 

278. Pear tree showing recently blighted limbs. At A', dead blight of the 

preceding year 362 

279. Pear blight on apricot in Washington, D. C 363 

280. Blighting pear leaves collected in Washington, D. C. May, 1915 364 

281. Shoot of Clapp's Favorite pear inoculated 5 days with a pure culture of 

Bacillus amylovorus 366 

282. Apple blight canker (Waite's hold-over blight). Spring condition — 

extruding bacteria 367 

283. A detail above figure 282 enlarged six times to show bacterial ooze 

from a limb 368 

284. Inoculated green pear fruit rotted by Bacillus amylovorus. It shows 

stomatal ooze in many places 369 

285. Blighting pear petiole. A detail from figure 280 showing the copious 

bacterial ooze from many stomata 370 

286. Cavities in a pear shoot due to Bacillus amylovorus. Only the cortical 

parenchyma is attacked 371 

287. A detail from figure 286 showing the bacteria. Inoculation of 1915 

on a young shoot of Clapp's Favorite. Time, 5 days 372 

288. Rods of Bacillus amylovorus as ordinarily seen in disintegrating tissues of 

pear fruit (fig. 284) 373 

289. Texas pear orchard destroyed by blight 375 

290. A, B. Pear trees in Maryland orchard, showing recovery from blight 

due to tree-surgery 376 

291. Flagellate rods of Bacillus amylovorus 377 

292. Surface and buried colonies of Bacillus amylovorus from agar-poured 

plates: .4, 1905; B, 1915 378 

293. A, B. Surface and buried colonies of Bacillus amylovorus on agar plates 

photographed by transmitted light; A, direct light; B, oblique light. . 379 

294. Buried and surface gelatin colonies of Bacillus amylovorus after 3 days 

at 21° C 381 

295. Action of Bacillus amylovorus on milk in the closed end of a fermentation 

tube 382 

296. Inoculated browned blighting pear shoot, showing beads of bacterial 

ooze. Variety "Blight-proof " 383 

296*. Pyrus ussuriensis Maxim., a species resistant to fire-blight 388 



LIST OF ILLUSTRATIONS XXV 

Page 

297. Tumor-bearing olive branches from Genoa, Italy 390 

298. Olive tubercles due to Bacterium savastanol EFS — pure culture in- 

oculations of 1903. Washington, D. C 391 

299. Olive branch showing dwarfing and death of terminal (inoculated) 

shoot and new surface infections below 391 

300. Young olive tree showing result of inoculating Bacterium savastanoi 

(at X) 392 

301. A detail from figure 300, about '^i natural size 393 

302. Cross-section of a young cheesy olive tubercle, slighth' magnified 393 

303. Like figure 302 but more highly magnified 395 

304. Channel of infection (X) in an olive petiole. Low power 396 

305. A detail from figure 304 at X, showing the bacteria. Highly magnified 397 

306. Cross-section of an olive twig at the level of a small tubercle which is 

composed chiefly of bark-cell proliferations 398 

307. Section oi small tubercle on under surface of an olive leaf 399 

308. Luigi Savastano. Photograph made in Naples at time he was studying 

olive tubercle (1897 to 1899) 400 

309. Flagellate rods of Bacterium savastanoi 401 

310. Agar surface and buried colonies of Bacterium sarasta>toi 402 

311. Surface colony of Bacterium savastanoi on gelatin 37 days. Year 1908. 

Enlarged 403 

312. Gelatin surface and buried colonies of Bacterium savastanoi. Year 1910. 

Enlarged 405 

313. Ringed surface colony of Bacterium savastanoi on + 10 peptone beef 

gelatin with 1 per cent dextrose. Enlarged 407 

314. Agar-poured plate of Bacterium savastanoi, ^-2 insolated (on ice) 30 

minutes with killing effect 408 

315. Inoculated crown gall on hop due to Bacterium tumefaciens Smith and 

Townsend 413 

316. Dwarfing effect of crown-gall on sugar-beet — inoculated 37 days 414 

317. 1. Crown gall on ho]) — Washington State. 2. Crown gall on rose — 

New Jersey. 3. Crown gall on apple limb — North Carolina. 4. 
Grafted crown gall on a pear seedling — Washington, D. C 415 

318. 1. Inoculated crown gall on yellow Paris daisy — 1 month la. Inoculated 

crown gall on yellow Paris daisy: one infected needle-prick; sterile 
pricks above. 2. Inoculated crown gall on peach — time, 5 months. 
2a. Ditto, time 18 days, 1907. 3. Inoculated crown gall on apple stem. 
4. Inoculated crown gall on grape. Time, 44 days 416 

319. 1. Inoculated crown gall on radish. 2. Paris daisy showing primary 

(inoculated) stem-tumor (at X); and 3 secondary (leaf") tumors — 
73 days. 3. Crown gall on Paris daisy, time 10 months. A new 
tumor developed below the sloughed old one. Stem dead. 4. 
Secondary crown gall on sunflower due to proliferation from a tumor- 
strand, primary tumor in the disk. 1915 5. Crown gall on Paris 
daisy. Section of stem between tumors showing a large tumor- 
strand. 6. Crown gall on Paris daisy. Section of stem between 
tumors showing 3 tumor-strands; also secondary tumors growing 

out of the leaf stubs 418 

■320. Crown gall on sugar beet — inoculated 3 months. Tissues sound 420 



XXVI LIST OF ILLUSTRATIONS 

Page 

321. Inoculated crown gall on sugar beets, showing necrosis 422 

322. Natural crown gall on willow limbs — from South Africa 423 

323. Tumor-strand in cross-section (in daisy stem near pith) — magnified. 

From an inoculated plant 424 

324. 1. Radial longitudinal section of leaf-ti'ace of Paris daisy, showing 

the normal anatomy, pitted vessels at right; spirals at left. 2. 
Radial longitudinal section of leaf-trace of inoculated Paris daisy, 
showing the interpolation of a tumor-strand — magnified. 3. Tumor 
strand in cross-section (stem of an inoculated Paris daisy), showing 
large cells with big nuclei. 4. Cross-section of stem of an inoculated 
Paris dais}^ with tumor-strand showing immature tracheids, develop- 
ing therefrom 425 

325. Crown gall tumor-strand in cortex of an inoculated Pelargonium 427 

326. Like figure 327, sub 4, but tumor full-grown and ruptured to surface 

of daisy leaf. Stem structure very distinct 428 

327. 1. Very early stage of crown gall on inoculated daisy stem. 2. Longi- 

tudinal section of unruptured small secondary tumor in petiole of an 
inoculated daisy. 3. Part of sub 2 enlarged. 4. Cross-section showing 
stem structure of secondary tumor in a daisy leaf — the tumor was 
still developing 429 

328. Lignin out of place in crown gall, i.e., deposited on walls of 3 large 

parenchyma cells 430 

329. Vascularized crown gall in bark parench3niia of Paris daisy induced by 

shallow needle-prick inoculations 431 

330. Inoculated crown gall on white Paris daisj' — showing a killed branch. 

Time, 7 months 432 

331. Nucleated cells of crown gall of daisy with bodies formerly identified 

as the bacteria in place. Gold chloride impregnation 433 

332. a, h. Crown gall on the daisy showing mitochondrial (?) rods formerly 

identified as Bacterium iumcfaciens within the cells. One rod branched 434 

333. Inoculated crown-gall teratoma on cauliflower, enlarged. Also side view- 

natural size 435 

334. Inoculated crown-gall teratoma on Ricinus communis (castor oil plant). 

Plant badly dwarfed. Normal stem of same age at right 436 

335. Tobacco crown gall bearing flower buds as result of a pure culture 

inoculation of Bacterium tumefacien,s 437 

336. A, B. Inoculated leafy crown galls on tobacco internodes 439 

337. Natural crown-gall witch-broom on Dianthus caryopbyllua (the car- 

nation) 440 

338. Inoculated crown-gall teratoma on hothouse geranium (Pelargonium).. 442 

339. A, B. Sections of an internodal tobacco teratoma 443 

340. A, B. Inoculated tobacco crown-gall teratomas showing development 

of shoots from leaves 444 

341. Crown-gall tumors and malformation on inoculated tobacco produced 

with the carnation isolation of Bacterium tumefaciens (fig. 337) 447 

342. Delayed crown-gall teratoma on inoculated orange stem 448 

343. Inoculated, slow-growing, hard, crown-gall teratoma on mango 450 

344. A. An embryomatous portion of the mango tumor (fig. 343) enlarged. 

B. Crown gall in center of an inoculated young orange fruit 452 



LIST OF ILLUSTRATIONS XXVll 

Page 

345. A detail from figure Mi B 454 

346. Like figure 345 but from another part of the same locule and further 

enlarged. Normal tissue at right. Disoriented tumor cells at left. 455 

347. Inoculated crown-gall teratoma on sugar beet 456 

348. Inoculated crown gall on top of garden balsam showing root.s develoijing 

from the tumors (hairy root) 458 

349. Flagellate rods of Bacterium tumefaciens. Organism from hop 459 

350. a, b. Y-shaped bodies of Bacterium tumefaciens from a culture treated 

with acetic acid 460 

35L Agar surface and buried colonies of Bacterium tumefaciens isolated from 

the Rose (colony P) 461 

352. Agar surface and buried colonies of Bacterium tumefaciens isolated 

from the Hop 462 

353. Inoculated crown gall of Paris daisy showing structure of the tumor 

(spindle cells) 464 

354. Structure of tumor tissue in crown gall on Paris daisy. Another part 

of figure 353 (round tumor cells) 465 

355. Margin of inoculated crown gall in sunflower pith showing l)oth crushing 

and invasion. Cells of tumor tissue disoriented 467 

356. Crown gall in gland of inoculated Ricinus communis. Epidermis 

involved and dividing 470 

357. Chestnut wood injected with lithium carbonate showing ingrowths 

(tyloses) in vessels 478 

358. 359. Cross-section and longitudinal section of tyloses in vessels of 

chestnut wood. Enlarged 479. 480 

360. Chestnut bark showing islands of wood (at 6) due to injection of lithium 

carbonate — a general view .* 481 

361, 362. Island of wood in chestnut bark. Enlarged 482, 484 

363. Cabbage pith showing effect of injecting sodium bicarbonate 485 

364 Tumors in Ricinus stem produced by vapor of monobasic ammonium 

phosphate 486 

365. Tumors on cauliflower leaf j^roduced by dilute vapor of ammonia 

water 487 

366. Acetic acid tumors on under surface of a cauliflower leaf. 1916 488 

367. Same as figure 366 but produced in 1918 490 

368. 369, 370, 371. Cross-sections of three acetic acid cauliflower tumors at 

different levels 491, 492, 493, 494 

372. Longitudinal section of an acetic acid cauliflower tumor 495 

373. 1-3. Formaldehyde tumors on cauliflower. 4. Section of the same 497 

374. 375, 376. Formic acid tumors on cauliflower leaves 498, .499, 500 

377. Structure of formic acid tumors on cauliflower leaves 501 

378. Photographs showing slight sub-epidermal injury preceding acetic acid 

tumors on cauliflower 503 

379. Harvey's frost tumors on a cabbage leaf 504 

380. 1, 2. Structure of Harvey's frost tumors on cabbage leaves 505 

381. Mottling of cauliflower leaf after exposure to vapor of ammonia water — 

it precedes tumor formation 506 

382. A, B. Wolf's sand blast intumescences on cabbage leaves 507 

383. Cross-section of \'ermont tomato leaf showing oedema 508 



XXVlll LIST OF ILLUSTRATIONS 

Page 

384. Atkinson's experimental intumescences on tomato 509 

385. A. Lenticel proliferations (semi-asphyxiation tumors) on Ficus clastica 

(rubber tree). B. Vertical section of the same, enlarged 512 

386. 1, 2. Lenticel proliferations (semi-asphyxiation tumors) on Morus alba 

(white mulberry). 3, 4. Untreated parts of same branch 514 

387. Vertical section of one of proliferating lenticel tumors shown in figure 386 516 

388. A, B. Proliferated (semi-asphyxiated) and normal lenticel of Olea 

europeoB (common olive) 517 

389. A, B. Proliferated (semi-asphyxiated) and normal lenticel in Begonia. . 518 

390. A, B, C. Tumors which formed accidentally on the sterile cut surfaces 

under the cork layer of a raw block of a potato tuber in a sealed tube 520 

391. Structure of the tumor at X on figure 390 521 

392. Block of pared sterile raw potato with small tumors experimentally 

duplicating figure 390 522 

393. Tumors on top and bottom of a block of pared sterile raw potato growing 

in a sealed tube. Cork layer ruptured 523 

394 Blocks of raw potato showing development of callous tissue and small 

tumors in sealed tubes at room temperature 524 

394*. Well developed intumescences on silver spotted begonia due to paint- 
ing with petrolatum in 1920 525 

395. Tumors on pared sterile surface of Early Rose potato tuber grown in a 

sealed tube on wet cotton 526 

396. Sterile cut surface of Early Ro.se potato tuber, showing a continuous 

series of tumors arising from the cambium and others not originating 

in the cambium 527 

397. Irish Cobbler potato showing tumors bursting through the normal skin of 

the tuber. Grown in a sealed tube on wet cotton 528 

398. Irish Cobbler from same series as figure 397. Tumors rupturing widely. 529 

399. General view of figures 397 and 398 showing method of treatment 530 

400. Intumescences (hyperplasias) developing under stomata on potato 

shoots in saturated air at high temperatures in bright light 532 

401. Like figure 400 but the basal intumescences fused and widely ruptured 

forming a collar 533 

402. Shoot of White McCormick potato grown in a sealed tube in the dark 

in saturated air at room temperatures — base covered with intvnn- 
escences, tip dying 534 

403. Sprouts shown in figure 399, enlarged X 5. Intumescences numerous. 535 

404. A, B. Same series as figure 403, but intumescences further ruptured. . . . 536 

405. Front and back view of a stunted swollen potato shoot exposed at 28° 

to 35° C. and covered with intumescences. From a pared sterile 
block in one of the sealed tubes: Effect of moist confined air, increased 
QO, and decreased oxygen 537 

406. Pared sterile blocks of Early Ohio potato in sealed tubes in the dark at 

23°-25° C, bearing shoots covered with intumescences ruptured and 
unruptured. Tips of roots and shoots asphyxiated 538 

407. Hyperplasia under a stoma on a potato shoot (fig. 406). Medium 

magnification 539 

408. Stages of growth in a tumor (teratoma) which developed from the 

surface of a pared sterile block of potato in a sealed tube at room 
temperature. Shoots covered with small hyperplasias 540 



LIST OF ILLUSTRATIONS XXIX 

Page 

409. Small intumescences on potato stems abundantly supplied with water 

but top destroyed by Bacillus phytophthorus 542 

410. Same as figure 409, but further enlarged and from the other side of the 

stem 544 

411. Intumescences (hyperplasias) on potato shoots which are dwarfed for 

lack of water — tissues full of sugar, starch, acids and oxydizing 
enzymes, base of shoots tumefied 546 

412. Vertical section through intumescences under stomata on a potato shoot 

(see figure 411). Tissue gorged with starch 547 

413. Surface view of hyperplasias on potato shoots from figure 411. Stoma 

central and wide open over each one 548 

414. A. B. Structure of a very young nematode tumor with the young worms 

in place. Shows both hypertrophy (giant cells) and hyperplasia. . . . 550 

415. Structure of a polythalamous gall on Rosa rubiginosa (the sweetbriar) 

leaves of an imusual type, i.e., somewhat resembling the calyx lobes. 552 

416. Magnified section of a part of fig. 4155 with two attached leaves 5.54 

417. Monothalamous cynipid gall from an oak tree {Quercus prinus ?): 

leaves of an unusual type, i.e., resembling those of the willow-leaved 
oak 556 

418. A. B. Enlarged views of the oak gall with some of the linear leaves 

removed 557 

419. Stained section of the center of fig. 418B magnified 25 times 559 

420. Side wall of fig. 419 further enlarged to show its structure 560 

421. Outer one-half of top wall of fig. 419 561 

422. Tangential section of the oak gall passing through the middle part of 

the wall — small cells of the hyperplasia in the middle, larger ones at 
either side, i.e., nearer the surface and farther from the larval ex- 
cretions 562 

423. Tangential section of the wall of the oak. gall showing, in the middle, 

the deep staining hypertrophied inner nutritive layer 564 

424. A. B. C. Cells of the hypertrophied inner layer, some containing more 

than one nucleus. Highly magnified 565 

425. Ordinary appearance of Begonia phijllomaniaca 576 

426. Non-proliferous and proliferous shoots of Begonia phyllomaniaca 577 

427. Enlarged central part of a dwarfed proliferous leaf showing that the 

proliferations are not restricted to the main veins 578 

428. A, B. Structure of leaf buds of Begonia phyllomaniaca; C. Plant grown 

from an adventive shoot on a leaf blade; D. Enlarged view of stem- 
lenticels and glands 580 

429. A, B. Proliferation on shoot and leaf stalk of Plant No. 1, first series, 

front and back view; C. Proliferation and beginning cork formation on 

a branch of the same 582 

430. 431. Dwarfed proliferous leaf of No. 1, first series, and next four leaves 

above it 584, 585 

432. Plant No. 6, first series, showing proliferous leaves and internodes with 

non-proliferous ones above and below 586 

433. Plant No. 6, first series, showing proliferous upper face of leaf blade Z 

and nearly smooth face of Z" next below it 588 

434. (1) Lower (free) and (2) middle (leafy) internodes of No. 6, first series: 



XXX LIST OF ILLUSTRATIONS 

Page 
(3) Branch of No. 1, first series showing cork formation in stimulated 
part 589 

435. Plant No. 8, first series, showing old and new proliferations on the main 

axis 591 

436. A. Plant No. 8, first series, third branch, showing proliferous and 

non-proliferous internodes; B. Plant No. 9, first series, whole of main 
axis with two stimulated proliferous internodes; C. Shoot arising from 
a trichome 592 

437. Plant No. 3, second series, main axis, showing dwarfing of proliferous 

leaf (M), adventive shoots on middle internodes, cork formation 
(Ck), etc 594 

438. Plant No. 9, second series, petiole and upper face of leaf M, blade 

proliferations mostly from the midribs 596 

439. A. Plant No. 9, first series, part of an internode enlarged with cork at 

C; B, Plant No. 9, second series, petiole enlarged. Both very pro- 
liferous 598 

440. A. Plant No. 10, second series, main axis, contrasting, especially, big 

proliferous and non-proliferous leaves; B. Cross-section of a petiole- 
trichome showing a bud arising from it 602 

441. Plant No. 10, second series: middle back part (center) of proliferous 

leaf blade J of fig. 440, enlarged 604 

442. Plant No. 10, second series, middle leaf, from a branch showing effect of 

vicmity of midribs on number and size of the adventive shoots 605 

443. Plant No. 15, second series, main axis, upper face of leaf-blade L and 

its 'petiole, full of adventive shoots 606 

444. 1, 2. Plant No. 18, second series, main axis, upper and lower part of the 

proliferous internode, enlarged 608 

445. Plant No. 1, third series of dried cuttings. Ck, cork; Tp, topmost 

small leaf when cutting was made; A', fallen leaf; Y, proliferous 
leaf; St, stipules. Proliferous leaf Y is dwarfed 610 

446. Plant No. 1, sixth series of cuttings, third, fourth and fifth leaves from 

the top. Stimulated proliferous leaf not dwarfed. 611 

447. Parts of leaves enlarged showing leafy shoots originating from the edge 

of knife wounds and needle pricks 613 

448. A. Shoots arising from knife-slit in lower surface of a midrib; B, effect 

of dwarfing on limitation of production of shoots from edges of wounds; 

C, two abnormal fused leaves from an adventitious shoot 614 

449. 1, 2. Petioles developing adventive shoots from trichomes. 3. Stimu- 

lated internodes which have developed adventitious shoots and cOrk 
formation. (PI. No. 6, first series.) 4. Same as 1 and 2. 5. Midrib- 
trichomes developing shoots 616 

450. A, B, C. Further evidence of the development of shoots from petiole 

hairs 617 

451. A. Cross-section of a leaf and, B, cross-section of an internode, showing 

superficial origin of the adventive shoots 618 

452. Abnormal leaves from adventive shoots: also stem-glands (x, y) giving 

rise to shoots 620 

453. Like 451A but an earlier stage of shoot-development from the epidermal 

region {x, y) 621 



BACTERIAL DISEASES 
OF PLANTS 



PART I 



A CONSPECTUS OF BACTERIAL DISEASES OF 

PLANTS 

All our knowledge of these diseases has come within a gen- 
eration. It began forty years ago with the announcement 
of the bacterial origin of pear blight by Professor T. J. Bur- 
rill of the University of Illinois (See Frontispiece), w^ho has but 
recently passed away.^ During the first half of this period 
progress w^as very slow and doubt universal, especially in Europe. 
In the early study of these diseases a few men were far in advance 
of their generation, as always happens when a new science un- 
folds. Photographs of the leading workers of that period, all 
of whom are still living, are shown in Fig. 1. All w^ere made at 
that time with exception of Savastano's which reached me too 
late and is shown separately as Fig. 308. 

It is now twenty-four years since I ventured the statement, ^ 
that "there are in all probability as many bacterial diseases 
of plants as of animals." This statement w^as received with 
much skepticism, not to mention active opposition, but tirhe 
has more than borne out my statement, and there is now^ no 
one left to dispute it. To-day I will venture another and 
broader generalization, to wit: It appears likely that event- 
ually bacterial diseases will be found in every family of plants, 
from lowest to highest. This prediction is based on the fact 
that although the field is still a very new^ one, with no workers 
in most parts of the world, such diseases have been reported 

1 Born in Massachusetts, April 25, 1839; deceased in Illinois, April li, 1916. 

2 Am. Nat., vol. 30, p. 627. 1896. 

1 



BACTERIAL DISEASES OF PLANTS 




Savastano 
Arthur 



Fig. 1. 
Wakker 



Cavara 
Waite 



CONSPECTUS : INTRODUCTION 




Fig. 2. — Fruit, fruit-stalk, and leaves of mango attacked by the South African 
bacterial disease. {After Ethel M. Doidge.) 



BACTERIAL DISEASES OF PLANTS 



from every continent, and are already known to occur in plants 
of one hundred and fifty genera distributed through more than 
fifty famihes. 

DISTRIBUTION 

Following Engler's arrangement, I will list these families 
that you may see how wide is the distribution of bacterial 
diseases in plants and how utterly wrong were those who said 
that there were no such diseases, and also those who conceded 
a little but said that they were very rare and restricted to the soft 
underground parts of a few bulbous and tuberous plants, and 
generally preceded by fungi (German writers and their follow- 
ers). In this list, I have included only the flowering plants, 
but some of the cryptogams are also subject to bacterial attack. 
The number following the family name indicates the number 
of bacterial diseases known within the limits of the family. 
The total of the figures, however, will not give the number of 
bacterial parasites, because some of the diseases overlap. 

TABLE 1 

SHOWING THE FAMILIES OF FLOWERING PLANTS ARRANGED SERIALLY FROM LOWEST 
TO HIGHEST. THOSE CONTAINING GENERA SUBJECT TO BACTERIAL DISEASES 
ARE UNDERSCORED, AND WHEN SEVERAL DISEASES HAVE BEEN RECOGNIZED 
THEIR NUMBER IS ALSO GIVEN • 



1. Cycadaceae 

2. Ginkgoaceae 

3. Taxaceae 

4. Pinaceae 2 

5. Gnetaceae 

6. Typhaceae 

7. Pandanaceae 

8. Sparganiaceae 

9. Potamogetonaceae 

10. Naiadaceae 

11. Aponogetonaceae 

12. Scheuchzeriaceae 

12. Juncnginaceae 

13. Alismaceae 

14. Butomaceae 
1.5. Vallisneriaceae 

1,5. H ydrocharitaceae 

16. Triuridaceae 

17. Poaceae 



17. Gramineae 14 

18. Cyperaceae 

19. Phoenicaceae 

19. Palmae 

20. Cyclanthaceae 

21. Araceae 

22. Lemnaceae 

23. riagellariaceae 

24. Baloskionaceae 

24. Restionaceae 

25. Centrolepidaceae 

26. Mayacaceae 

27. Xyridaceae 

28. Eriocaulaceae 

29. Rapateaceae 

30. Bromeliaceae 

31. Cominelinaceae 

32. Pontederiaceae 

33. Philvdraceae 



34. Juncaceae 

35. Stemonaceae 

36. Melanthiaceae 

37. Liliaceae 3 

38 .Convallariaceae 

39. Smilacaceae 

36. ] 

37. I 
38. 



Liliaceae 



39. 



J 



40. Haemodoraceae 

41. Amaryllidaceae 

42. Velloziaceae 

43. Taccaceae 

44. Dioscoreaceae 

45. I ridaceae 

46. Musaceae 

47. Ziii^iheraceae 

48. Cannaceae 



conspectus: distribution 



49. Marantaceae 

50. Burmanniaceae 

51. Orchidaceae 7 (?) 

52. Casuarinaceae 

53. Saururaceae 

54. Piperaceae 

55. Chloranthaceae 

56. Salicaceae 2 

57. Myricaceae 

58. Balanopsidaceae 

59. Leitneriaceae 

60. Juglandaceae 2 

61. Betulaceae 

62. Fagaceae 2 

63. Ulmaceae 

64. Moraceae 

65. Urticaceae 5 
6(3. Proteaceae 

67. Loranthaceae 

68. Myzodendraceae 

69. Santalaceae 

70. Grubbiaceae 

71. Opiliaceae 

72. Olacaceae 

73. Balanophoraceae 

74. Aristolochiaceae 

75. Rafflesiaceae 

76. Hydnoraceae 

77. Polygonaceae 2 

78. Chenopodiaceae 5 

79. Ainaranthaceae 

80. Nyctaginaceae 

81. Batidaceae 

82. Theligonaceae 

82. Cynocrambaceae 

83. Phytolaccaceae 

84. Aizoaceae 

85. Portulacaceae 

86. Basellaceae 

87. Silenaceae 

87. Cari/ophyllacene 2 

88. Nymphaeaceae 

89. Ceratophyllaceae 

90. Trochodendraceae 

91. Ranunculaceae 

92. Lardizabalaceae 

93. Berberidaceae 

94. Menispermaceae 



TABLE I.— (Continued) 

95. Magnoliaceae 

96. Calycanthaceae 

97. Lactoridaceae 

98. Annonaceae 

99. Myristicaceae 

100. Gomortegaceae 

101. Monimiaceae 

102. Lauraceae 

103. Hernandiaceae 

104. Papaveraceae 2 (?) 

105. Brassicaceae 

105. Cruciferae 5 

106. Tovariaceae 

107. Capparidaceae 

108. Resedaceae 

109. Moringaceae 

110. Sarraceniaceae 

111. Nepenthaceae 

112. Droseraceae 

113. Podostemonaceae 

114. Hydrostachyaceae 

115. Crassulaceae 

116. Penthoraceae 

115 1 

> CrassuJarrop 

116. / 

117. Cephalotaceae 

118. Sa.xifragaceae 

119. Hydrangeaceae 

120. Escalloniaceae 

121. Grossulariaceae 2 

118. ] 

119. 

120. 

121. ] 

122. Pittosporaceae 

123. Brunelliaceae 

124. Cunoniaceae 

125. Myrothamnaceae 

126. Bruniaceae 

127. Hamamelidaceae 

128. Platanaceae 

129. Crossosomataceae 

130. Rosaceae 

131. Malaceae 

132. Amygdalaceae 

130. ] 

131. i Rosaceae 7 

132. I 



Saxifragaceae 



133. 


Connaraceae 


134. 


Mimosaceae 


135. 


Caesalpiniaceae 


136. 


Krameriaceae 


137. 


Fabaceae 


134. 


. Lequminosae 7 


135. 
136. 




137. 


138. 


Geraniaceae 2 


139. 
140. 


Oxalidaceae 
Tropaoolaceae 3 


141. 


Linaceae 


142. 


Humiriaceae 


143. 


Ervthroxvlaceae 


144. 


Zygoph.vllaceae 


145. 
146. 


Cneoraceae 
Rutnceae 4 


147. 


Simaroubaceae 


148. 


Balsam eaceae 


148. 
149. 


Burseraceae 
Meliaceae 


150. 
151. 


IMalpighiaceae 
Trigoniaceae 


152. 


Vochvaceae 


152. 


Vochysiaceae 


153. 


Tremandraceae 


154. 
155. 
156. 


Pol.vgalaceae 
Difhapetalaceae 
Eiiphorliiaceae 2 


157. 


Callitrichaceae 


158. 


Buxaoeae 


159. 


Coriariareae 


160. 


Empetraceae 


161. 

162. 


Limnanthaceae 
Anacardiaceae 


163. 


Cyrillaceae 


164. 


Pentaph.vlacaceae 


165. 


Corynocarpaceae 


166. 
167. 


Aquifoliaceae 
Celastraceae 


168. 


Hippocrateaceae 


169. 


Stackhousiaceae 


170. 


Staphyleaceae 


171. 


Icacinaceae 


172. 


Aceraceae 


173. 


Aesculaceae 



BACTERIAL DISEASES OF PLANTS 



173. Hippocasianaceae 

174. Sapindaceae 

175. Sabiaceae 

176. Bersamaceae 

176. Melianlhaceae 

177. Impatientaceae 
177_ Balsaniinaceae 2 

178. Rhamnaceae 

179. Vitaceae 4 

180. Elaeocarpaceae 

181. Schizolaenaceae 

181. Chlaenaceae 

182. Gonystylaceae 

183. Tiliaceae 

184. Malvaceae 3 

185. Tripiochitonaceae 

186. Bombacaceae 

187. Sterculiaceae 

188. Scytopetalaceae 

189. Dilleniaceae 

190. Eucryphiaceae 

191. Ochnaceae 

192. Caryocaraceae 

193. Marcgraviaceae 

194. Quiinaceae 

195. Theaceae 

196. Hypericaceae 

197. Clusiaceae 
196. 
197. 

198. Dipterocarpaceae 

199. Elatinaceae 

200. Frankeniaceae 

201. Tamaricaceae 

202. Fouquieriaceae 

203. Cistaceae 

204. Bixaceae 

205. Cochlospermaceae 

206. Koeberliniaceae 

207. Canellaceae 

208. Violaceae 

209. Flacourtiaceae 

210. Stachyuraceae 

211. Turneraceae 

212. Malesherbiaceae 

213. Passifloraceae 

214. Achariaceae 

215. Papayaceae 

215. Caricaceae 

216. Loasaceae 

217. Datiscaceae 



Guttiferae 



TABLE I.— {Continued) 

218. Begoniaceae 2 

219. Ancistrocladaceae 

220. Cactaceae 

221. Geissolomaceae 

222. Penaeaceas 

223. Oliniaceae 

224. Thymelaeaceae 

225. Elaeagnaceae 

226. Lythraceae 

227. Blattiaceae 

227. Sonneratiaceae 

228. Crypteroniaceae 

229. Punicaceae 

230. Lecythidaceae 

231. Rhizophoraceae 

232. Combretaceae 

233. Myrtaceae 

234. Melastomataceae 

235. Onagraceae 

236. Trapaceae 

236. Hydrocaryaceae 

237. Haloragidaceae 

237. Halorrhagidaceae 

238. Cynomoriaceae 

239. Araliaceae 2 

240. Apiaceae 

240. Urnhelliferae 4 

241. Cornaceae 

242. Clethraceae 

243. Pyrolaceae 

244. Monotropaceae 

> Pyrolaceae 

245. Lennoaceae 

246. Ericaceae 

247. Vacciniaceae 

\ Ericaceae 

247. / 

248. Epacridaceae 

249. Diapensiaceae 

250. Theophrastaceae 

251. Myrsinaceae 

252. Primulaceae 

253. Plumbaginaceae 

254. Sapotaceae 

255. Diospyraceae 

255. Ebenaceae 

256. Styracaceae 

257. Symplocaceae 

258. Oleaceae 3 



259. Salvadoraceae 

260. Loganiaceae 

261. Gentianaceae 

262. Menyanthaceae 

2C2 ( Gentianaceae 

263. Apocynaceae 

264. Asclepiadaceae 

265. Convolvulaceae 

266. Cuscutaceae 

> Convolvulaceae 

266. j 

267. Polemoniaceae 

268. Hydrophyllaceae 

269. Boraginaceae 

270. Verbenaceae 

271. Menthaceae 

271. Labiatae 

272. Nolanaceae 

273. Solanaceae 10 

274. Scrophulariaceae 

275. Bignoniaceae 

276. Pedaliaceae 

277. Martyniaceac 

278. Orobanchaceae 

279. Gesneriaceae 

280. Columelliaceae 

281. Pinguiculaceae 

281. Lentibulariaceae 

282. Globulariaceae 

283. Acanthaceae 

284. Myoporaceae 

285. Phrymaceae 

286. Plantaginaceae 

287. Rubiaceae 

288. Caprifoliaceae 

289. Adoxaceae 

290. Valerianaceae 

291. Dipsacaceae 

292. Cucurbitaceae 3 

293. Campanulaceae 

294. Goodeniaceae 

295. CandoUeaceae 

296. Calyceraceae 

297. Cichoriaceae 

298. Ambrosiaceae 

299. Asteraceae 

297. ] 

298. \ Comyositae 6 

299. I 



conspectus: distribution / 

The widest gaps, it will be observed, are bet\Yeen Cruciferae 
and Rosaceae and between Hypericaceae and Begoniaceae, but 
these I beUeve represent nothing more than lack of knowledge.^ 

I give below a list of genera within the limits of which one 
or more species are now said to be subject to attack. Many 
of these genera contain plants of great economic importance. 
Where I have some personal knowledge of the subject I have 
italicized the genus name, and in what follows the reader will 
naturally expect me to draw illustrations principally from the 
diseases most familiar to me. 







TABLE 11 




SHOWING GENERA OF FLOWERING PLANTS SUBJECT TO DISEASES OF BACTERIAL OKI G 


Macrozamia 


Dendrobium 


Delphinium 


Trifolium 


Pinus 


Cattleya 


Papaver 


Medicago 


Hordeum 


Oncidium 


Brassica 


Arachis 


Dactylis 


Odontoglossum Raphanus 


Acacia 


Bromus 


Cypripedium 


Cheiranthus 


Prosopis 


Zea 


Phalaenopsis 


Matthiola 


Erythrina 


Setaria 


Vaydlla 


Ribes 


Geranium 


Andropogon 


Salix 


Amelanchier 


Erodium 


Avena 


Populus 


Sorbus 


Pelargonium 


Saccharum 


Juglans 


Eryobotrya 


Tropaeolum 


Secale 


Castanea 


Pyrus 


Chaetospermum 


Triticum 


Corylus 


Cydonia 


Fortunella 


Phleum 


Morus 


Prunus 


Citrus 


Poa 


Pouzolzia 


Rubus 


Lansium 


Agro-ptjron 


Protea 


Cratcegus 


Cedrela 


Cocos 


Cannabis 


Fragaria 


Manihot 


Oreodoxa 


Acalypha 


Rosa 


Hevea(?) 


Richardia 


Hum ulus 


Heteromeles 


Rici7i us 


Amorphophallus 


Ficus 


Dolichos 


Euphorbia 


Hyacirithus 


Hippocralea 


Lathyrus 


Mangifera 


Allium 


Rheum 


Indigofera 


Euonymus 


Lilium 


Polygonum 


Kraunhia (?) 


Impatiens 


Iris 


Atriplex 


Lupinus 


Vitis 


Ixia 


Spinacia 


Mucuna 


Gossypium 


Gladiolus 


Beta 


Phaseolus 


Malva 


Musa 


Amaranthus 


Cicer 


Sterculia 


Zingiber 


Dianlhus 


Vigna 


Elodea 


Canna 


Nelumbium 


Pisum 


Begonia 



^ Since this was written two bacterial diseases have been reported on Ribes 
and I have inoculated crown gall into Resedaceae and Crassulnceae. 



BACTERIAL DISEASES OF PLANTS 



TABLE 11.— {Co7Uinued) 



Opuntia 


Diospyros 


Nicotiana 


Ambrosia 


Eucalyptus 


Ligustrum 


Physalis 


Crepis (?) ' 


Oenothera 


Syringa 


Petunia 


Ageratum 


Aralia 


Olea 


Dot ura 


Chrysanthemum 


Hedera 


Fraxinus 


Calceolaria 


Lad uca 


Daucus 


Strychnos 


Sesatnum 


Blumea 


Pastinaca 


Nerium 


Pavetta 


Spilanthes 


Levisticum 


Symphytum 


Psycotria 


Pluchea 


Apium 


Tectona 


Benincasa 


Synedrella 


Arbutus 


Verbena 


Cucumis 


Calendula 


Vaccinium 


Coleus 


Cucurbita, 


Tragopogon 


Ardisia 


Salvia 


Citrullus 


Bellis 


Crispardisia 


Capsicum 


Sicyos 


Hdianthus 


Amblyanthus 


Solanum 


Echinocystis 


Aster 


Amblyanthopsis 


Lycopersicum 


Eclipta 





PERIOD OF GREATEST SUSCEPTIBILITY 



In certain diseases the brief seedling stage of the plant is 
the one most subject to attack, e.g., Stewart's disease of maize 
due to Aplanobacter stewarti, and brown rot of tomato and to- 
bacco due to Bacterium solanacearum, but many bacterial 
diseases of older plants are also rather strictly time-limited. 
In both groups it is a question of abundant immature tissue. 
To the latter class belong the numerous leaf-spots, fruit-spots, 
and blights, e.g., black spot on the plum and peach, due to 
Bacterium pruni, the fire-blight of the pear, apple, quince, etc., 
due to Bacillus amylovorus and the blight of the mango due to 
Bacillus mangiferce (Figs. 2, 3,). In such cases, so far at least 
as they occur in temperate climates, the disease appears in the 
spring and the greater part of it occurs during a brief period 
in the early summer, in which growth of roots, leaves and shoots 
is proceeding rapidly and there are many young and succulent 
parts. The cause of the disease may and often does remain on 
the plant over winter in a latent or semi-latent condition (walnut 
blight, pear blight, plum canker, etc.), but the active period is 
limited to three months, more or less, of actively growing weather 
in which developing tissues, subject to infection, are abun- 
dant. With the end of rapid growth and the hardening of the 



conspectus: period of greatest susceptibility 



9 



tissues in late summer and autumn, the disease is checked and 
disappears, or remains as a slow canker to appear again on other 
parts the following spring. It is a very instructive experiment 
to see, for example, inoculations of Bacillus amylovorus on ripen- 






'^^ 

^Tf 






k r 



f^0Si 



.^ ^w- 



v4^^ 



'^ 



V 



ItA 



n^ 4 



// 



Fig. 3. — Mango leaves show- 
ing bacterial spots. Specimens 
received from South Africa. 
(Courtesy of Miss Doidge.) 







Fig. 4. — West Indian coconut l:)ud- 
rot. The re flexed persistent dead fronds are 
typical. (After John R. Johnston.) 



ing fruits and shoots of the pear wholly fail toward the end 
of summer, which were eminently successful on the same trees 
at its beginning. The difference Id this case is not due to 
lessened virulence on the part of the organism, but to changes 
in the host-plant, making it non-susceptible. Similar changes 



10. BACTERIAL DISEASES OF PLANTS 

leading to non-susceptibility occur in the Japanese plum sub- 
ject to Bacterium pruni; the young fruits are very susceptible, 
the maturing fruits cannot be infected. In the destructive 
coconut bud-rot of the West Indies (Fig. 4) only the young 
"swords" and the undeveloped, sheathed, terminal bud are 
attacked by the bacteria. 

Other parasites, on the contrary, are able to attack, disin- 
tegrate and destroy matured tissues, such as the pith of cabbage 
stems, turnip roots, the ripened tubers of the potato, the well-de- 
veloped roots of sugar beets and of carrots, the bulbs of onions 
and hyacinths, full-grown melon and cucumber fruits. 

In both of these types the action of the parasite is expended 
chiefly on the parenchyma. Although in some cases (the plum 
disease, Appel's potato rot) there is more or less bacterial in- 
vasion of the local vessels, vascular occupation is not a special 
characteristic. 

In the typical vascular diseases the case is reversed. Here 
parenchyma is also destroyed, more or less, but the most con- 
spicuous and destructive action is on the vascular bundles, 
the hadrome vessels of which are occupied for long distances, 
to the death, or great detriment, of the whole plant. In maize 
attacked by Aplanobacter stewarti, it is not unusual, indeed one 
might rather say it is customary, to find the vessels of the stem 
filled with the bacteria continuously for a distance of 3 to 6 
feet from the point of infection, i.e., from the surface of the earth 
to the top of the full-grown plant. In cucurbits attacked by 
Bacillus tracheiphilus, in bananas attacked by Bacillus musae 
(Fig. 5) and in sugar-cane attacked by Bacterium vascularum the 
same thing occurs, and many of the vessels are filled solid with 
the bacterial slime to a distance of 8 or 10 feet from the place of 
infection. In such cases infection has taken place, generally, 
near the base of the plant which continues to grow for some 
weeks or months. 

Transitions, of course, occur. For example, Aplanobacter 
stewarti, is confined much more strictly to the vascular bundles 
of the maize stem than is Bacterium solanacearum to those of 
the tomato, potato, or tobacco stem, although it also is a vascu- 
lar parasite; that is, following infection of the vessels we do not 



conspectus: period of greatest susceptibility 11 




Fig. 5. — Cross-section of a banana fruit-stalk from Trinidad showing drops of 
the slime of Bacillus nmsoe Rorer oozing from the vascular bundles, some of which 
together with the drops are stained brown. X 3. 
Photo, by James F. Brewer. 



12 BACTERIAL DISEASES OF PLANTS 

find in the maize stems that extensive breaking down of the 
pith and bark into vast cavities which is so common, for example, 
in tobacco and tomato stems. 



WHAT GOVERNS INFECTION 

Within the plant we may suppose, from certain indications, 
that abundant juiciness is one of the factors governing the in- 
fection of immature tissues. To this may be added an abun- 
dant supply of well-adapted food and, in some cases, probably 
the absence of inhibiting substances, which may appear later. 
As parts approach maturity, the intercellular air-spaces become 
much larger and the water content becomes relatively less. 
Along with this, acids, sugars, proteids, amino-acids, etc., are 
consumed and converted into substances less well adapted to 
the needs of the meristem-parasites, if not wholly inimical. 
In young shoots of potato and tomato, or of pear and apple, 
as contrasted with old ones, or in the roots of carrots as .com- 
pared with the leaves, or in juicy carrots as compared with 
flabby ones, or in rapidly growing cabbages as compared with 
slow-growing ones, we know that there is an excess of water, and 
this alone appears to be sufficient to explain the difference in 
behavior of their respective parasites in old versus young parts. 
When, however, we come to ripening fruits, such as the pear 
and the plum, it would seem that they are still juicy enough to 
favor the growth of almost any bacterium ; we are forced, there- 
fore, to the hypothesis of chemical changes within the fruits 
to account for the failure of inoculations, and this throws some 
doubt on the preceding hypothesis. As a rule (there are striking 
exceptions), parasitic micro-organisms are rather sensitive to 
changes in their environment, e.g., to drying, exhaustion of food- 
supplies, multiplication of their own by-products, conversion 
of an easily assimilable substance into one less assimilable or 
actually harmful, appearance of esters, new acids, etc. But why 
speculate! Much additional experimenting must be under- 
taken before we shall have precise and full data. We are still 
largely in the observational stage and experiments are needed.^ 

1 In the above connection the following list of fruit acids may be of some use: 



conspectus: what governs infection 13 

The parasites of ripened tissues do not require so much 
water, are able to convert starch into sugar, or have a special 
liking for some other element of the plant tissue. 

Externally, a number of factors favor infection. One of 
these is excessive shade, either of clouds or of foliage, and an- 
other is high temperature. When these two factors are accom- 
panied by excessive rainfall, high winds, wet earth, and heavy 
dews, the conditions are ideal for the rapid dissemination and 
the destructive prevalence of a variety of bacterial diseases of 
cultivated plants. The bean blight due to Bacterium phaseoli, 
the angular leaf spot of cucumber due to Bacterium lachrijmans 
(Figs. 6 to 9) , the black spot and canker of the plum due to Bac- 
terium pruni, and the lark-spur disease due to Bacterium del- 
phinii, are all favored by heavy dews and by shade. In hot, 
wet weather in midsummer, pear blight due to Bacillus amy- 
lovorus often bursts out like a conflagration and sweeps over 
whole orchards. In warm, moist autumns bacterial diseases 
of the potato may destroy almost or quite the entire crop over 
extensive districts. 



Fruit Acids found 

Apple Malic only. 

Banana Probably malic only. 

Cantaloupe Malic none — probably all citric. 

Cherry Malic only. 

Cranberry Citric probably predominates — malic also present. 

Currant Citric probably predominates — malic sometimes pres- 
ent. 

Gooseberry Malic and citric. 

Peach Probably malic only. 

Pear Malic only in some varieties; citric probably predomi- 
nates in others with small amounts of malic. 

Persimmon Probably malic only. 

Plum Malic only. 

Pomegranate Probably all citric — no malic or tartaric. 

Quince Malic only — no citric. 

Raspberry (red) Probably citric only — malic, if present, in traces only. 

Watermelon Malic, no citric. 

Jour. Amer. Med. Assoc, vol. Ixix, No. 17, Oct. 27, 1917, p. 1433. 
Bigelow, W. D., and Dunbar, P. B.: The acid content of fruits, Jour. Industrial 
and Engineering Chem., 1917 (9, 762). 



14 



BACTERIAL DISEASES OF PLANTS 




Fig. 6. — Angular leaf-spot on cucumber due to stomatal infections produced 
by spraying-on Bacterium lachrymans Smith and Bryan. It shows breaking of 
tissue around old spots. Time, 12 days. 




Fig. 7. — Cucumber stem showing white bacterial film and cracks due to Bac- 
terium lachrymans. Inoculated by spraying May 6, 1915. Photographed May 20. 
X 14. 



conspectus: how infection occurs 



15 



HOW INFECTION OCCURS 

As I have already described elsewhere how infection oc- 
curs/ I will dwell on it here only for a moment, offering a few 
examples. 

The commonest way of infection is probably through wounds. 
In Italy, the olive tubercle due to Bacterium savastanoi has 
been observed to begin very often in wounds made by hail- 





FiG. S. Fig. 9. 

Fig. 8. — A beef peptone gelatin ( + 10) stab culture of Bacterium lachrymans 
after 12 days at 20°C. In the unliquefied part the colonies along the needle track 
are very small showing that it is aerobic. 

Fig. 9. — Two different illuminations of a small gelatin colony of Bacterium 
lachrymans to show the characteristic margin. X 14. 

stones. In South Africa, crown gall is said to be disseminated 
in the same way. In this country and also in Sumatra, Bac- 
terium solanacearum enters the plant more often than other- 
wise through broken roots. A tomato or tobacco plant with 

1 Smith, Erwin F.: "Bacteria in relation to plant diseases," Carnegie Inst. 
Washington, Publ. 27, Vol. 2, pp. 51-64, 1911. 



16 BACTERIAL DISEASES OF PLANTS 

unbroken roots will thrive in a soil deadly to one that has been 
root-pruned. I have myself observed this. We may suppose 
that substances attractive to the particular bacteria diffuse into 
the soil from the broken roots, following which they enter the 
plant. Resistant plants may be supposed to diffuse indifferent 
or repellant substances. All infections must be chemotactic. 

More interesting perhaps are those diseases which begin in 
natural openings, i.e., in places where the protective covering 
of the plant gives place to special organs such as nectaries, water- 
pores, and stomata. 

All the pome fruits subject to fire-blight are liable to blos- 
som infection. The bacteria multiply first in the nectaries of 
the flower and pass down into the stem by way of the ovary 
and pedicel. Blossom blight of the pear is a very conspicuous 
and common form of the disease, as everybody knows. Thou- 
sands of blighted blossom-clusters may be seen in any large 
orchard subject to this disease. Blossom-blight arises from 
''hold-over" blight (see Figs. 282 and 283), the visiting insects 
acting as carriers. 

In the black rot of the cabbage due to Bacterium campestre, 
the majority of the infections begin in the water-pores. These 
are grouped on the margins of the leaf at the tips of the ser- 
ratures. From this point the bacteria burrow into the vas- 
cular system of the leaf and so pass downward into the stem 
and upward into other leaves. 

In the black spot of the plum, due to Bacteriuin pruni almost 
or quite all of the leaf and fruit infections are stomatal. A 
large proportion of them are also stomatal in the leaf-spot of 
cotton due to Bacterium malvacearum, the leaf-stripe of sorghum 
and broom-corn due to Bacterium andropogoni, the leaf-spot of 
carnations due Bacterium woodsii, and other leaf-spots.^ 

TIME BETWEEN INFECTION AND APPEARANCE OF THE DISEASE 

As in animal diseases, the period of latency may be very 
short or surprisingly long. Some time must be allowed the 

1 The writer first called attention to stomatal infections in 1897, having demon- 
strated their existence experimentally. See "Bacteria in Relation to Plant 
Diseases," Vol. 2, pp. 39, 56, 57; Pls.^3, 4, and Figs. II, 12, 15, 16,^17. 



conspectus: period of incubation 17 

parasitic organism to multiply inside the plant before it does 
damage serious enough to be recognized externally as a dis- 
ease. This is the so-called ''period of incubation," during 
which the parasite is growing and its enzymes and toxins are 
becoming active. The microscope shows it to be present in the 
tissues, but the latter have yielded only a little in the immediate 
vicinity of the bacterial focus. This time is short or long 
depending on whether the parasite or the host has the first 
advantage. If the host is growing rapidly it may either en- 
tirely outstrip the parasite, or be only so much the more sub- 
ject to it. All depends on whether the parasite finds the initial 
conditions entirely suited to its needs, or by means of its secre- 
tions and excretions can quickly make them so, and conse- 
quently can from the start make a rapid growth, or must 
first slowly overcome obstacles of various sorts, such as in- 
hibiting acids and resistant tissues. The plant may show signs 
of infection within as short a time as one or two days after in- 
oculation (various soft rots), or it may be as long a time as one 
to two months before they appear (Cobb's disease of sugar-cane, 
Stewart's disease of sweet-corn). In the latter, infection gener- 
ally occurs in the seedling stage and the maize plant may be three 
months old and six feet tall before it finally succumbs. Of 
course, as in case of bacterial animal diseases, the greater the 
volume of infectious material, the shorter the time. I have 
seen many instances of that law. In general, the period of 
latency may be said to vary from one to three weeks (yellow 
disease of hyacinth, black rot of cabbage, black spot of plum, 
cucurbit wilt, pear blight, angular leaf-spot of cotton, sorghum 
leaf -stripe, etc.). The longest period of latency I have observed 
was two years. This was in crown gall on orange (see Fig. 342). 

RECOVERY FROM DISEASE 

Mention has already been made of the self-limited spot 
diseases and blights. As the actively growing season draws 
to a close such diseases cease their activity. 

Also in some plants well developed signs of vascular dis- 
ease may be suppressed (squash, maize, sugar-cane, etc.), or 



18 



BACTERIAL DISEASES OF PLANTS 




Fig. 10. — Two young tomato plants of a sensitive variety inoculated and 
wilting on leaf marked A', as a result of needle-pricks introducing the Sumatran 
strain of Bacterium solanacearum which had been exceedingly virulent but was'now 
losing its power to infect. These plants recovered and are shown in Fig. IL 



*P^?^ -^ 




Fig. 1L — Same as Fig. 10, but after 2V2 months. Plants recovered. The 
only part of the stem to develop incipient roots was the base near the^ inoculated 
leaf. Plants 2 feet 6 inches and 2 feet 11 inches high. Leaves not reflexed. 



conspectus: recovery from disease 



19 



remain in abeyance for a longer or shorter period, according to 
the varying fortunes of the host and the capabihties of the 
parasite. The tomato plants inoculated with Bacterium sol- 
anacearum (Medan iii) and photographed for Volume iii of 
''Bacteria in Relation to Plant Diseases/' 
(Plate 45 D), entirely outgrew the disease 
(Figs. 10 and 11), as did also certain sugar- 
canes (series vi) inoculated with Bacterium 
vascularum.^ Also, I have seen tomato 
plants recover only to develop a second and 
fatal attack of the vascular brown rot three 
months after the first attack, during which 
period they had made an extensive healthy- 
looking growth. 2 

Recovery from disease may depend on 
loss of virulence on the part of the para- 
site. This loss often occurs when bacteria 
are grown for some time on culture-media, 
and it occurs also in nature, but its cause 
is obscure; possibly it is due to oxidations. 
Practically nothing is yet known about 
acquired immunity on the part of the host 
plant. 

AGENTS OF TRANSMISSION 



These may be organic or inorganic. 
In many cases the plant itself harbors the 
parasite indefinitely, carrying it over from 
year to year on some portion of its growth 
(Pear blight, citrus canker, crown gall, etc.). 

Seeds, tubers, bulbs, grafts, or the 
whole plant may be responsible for the 
appearance of the disease the following 
year in the old localities, and through the 
agency of seedsmen, nurserymen, or whoever 
for outbreaks in regions hitherto exempt. 

1 Smith, Erwin F.: "Bacteria in relation^ to plant 
Washington, Publ. 27, Vol. 3, p. 33, 1914. 

2 Ibid., p. 179. 




Fig. 12. — Bacterial 
black chaff disease of 
wheat. Kansas, end of 
June, 1915. Black 
stripes on glumes and 
rachis. Kernels shriv- 
eled and also bacterially 
invaded. X 2}^. 

disseminates plants, 



diseases," Carnegie Inst. 



20 



BACTERIAL DISEASES OF PLANTS 



There is good reason to believe that the black rot of cabbage 
and Stewart's disease of sweet corn have been disseminated 
broadcast in the United States in recent years by ignorant and 
unscrupulous seedsmen. Both diseases are transmitted to seed- 
ling plants from the seed. The bacterial black chaff of wheat 
(Figs. 12 to 22) which is widely prevalent in Kansas, Iowa and 




Fig. 13. — A single yellow surface colony of Bacterium tranducens var. undu- 
losurn Smith, Jones and Reddy, the schizomycete causing black chaff of wheat. 
A glume isolation on +15 beef -peptone agar from No. 273, Dubois, Nebraska. 
Photographed by oblique, transmitted light to show internal wave-like markings, 
surface perfectly smooth. Plate poured July 9, 1917. Photo July 30, Temp. 
28°-32°C. X 10. 



other Western States is a seed-borne infection, and so is the very 
similar barley disease described by Jones, Johnson and Reddy. 
The yellow disease of hyacinths is carried in the bulb. Potato 
tubers from diseased fields may infect healthy fields. Apple 
grafts have transmitted crown-gall. Slightly infected trunks 
and limbs of trees (hold-over pear blight, walnut blight, canker 
of the plum) may infect shoots, leaves, blossoms, or fruits 



conspectus: agents of transmission 21 

the following season. The soil around the infected plant may 
serve, it is believed, for years as a source of infection to other 
species (crown gall), or to other individuals of the same kind 
(various leaf-spots). Occasionally, however, a parasite seems 
to die out of certain soils {Bacterium solanacearum, Bacillus 
tracheiphilus) . The pear blight organism probably dies as 
quickly in soils as it does in a majority of the blighted branches. 
Pear blight- or cucurbit wilt by soil-infection is not known. 

Among extraneous agents, wind and water have been sus- 
pected. I have never seen any clear indications of purely wind- 
borne infection, not even when contiguity seemed to invite it, 
but water and, of course, in driving rains, the wind, also, often 
carries parasites and furnishes conditions favorable to infection 
(citrus canker, angular leaf-spot of cotton, and bacterial canker 
of the tomato due to Aplanobacter michiganense) . Home has 
shown that the olive tubercle in California may be transmitted 
from the surface of diseased branches to sound branches by rain 
or dew (see Fig. 300). Honing, in the tobacco fields of Sumatra, 
has traced infection several times to the watering of plants 
from infected wells, and has cultivated the parasite from the 
water. I have discovered experimentally that to obtain in 
abundance several sorts of bacterial leaf -spots, e.g., those occur- 
ring on bean, cotton, peach, plum, carnation, larkspur, sorghum, 
geranium, etc., the leaves must be kept moist to the same extent 
they would be in case of prolonged dews or frequent light show- 
ers. In nature such conditions are necessary to enable the 
bacteria to penetrate the stomata and begin to grow. In case of 
water-pores, however, the plant itself furnishes the water neces- 
sary for infection, if the nights are cool enough, i.e., if the air 
remains near enough to saturation to prevent for some hours the 
evaporation of the excreted water from the leaf-serratures. 
Every plant with functioning water-pores awaits its appropriate 
bacterial parasite. The genus hnpatiens is a good example. I 
have looked for one on it in vain but I am sure it must occur. 

Man and the domestic animals, especially through the agency 
of the dung-heap, infallible repository of all sorts of discarded 
refuse, undoubtedly help to spread certain bacterial diseases 
of plants (potato rots, black rot of cabbage, etc.). 



22 



BACTERIAL DISEASES OF PLANTS 





\ 



Fig. 14.-Bacterial black chaff of wheat. Stalk bent double to show diseased 
(black-striped) upper part and sound (pale green) middle part. Wheat No. 268, 
collected June 28, 1917, on the Rhodes farm at Manhattan, Kansas. Photo- 
graphed July 3, 1917, by James F. Brewer, using a W. and W . panchromatic plate 
and a yellow color screen. 



conspectus: agents of transmission 



23 




■■ "wW.r.^rT^'^'.i. 



Fig. 15.— Montana spring wheat. Crop of 1917. Coll. iNo. 318. Diseased 
glumes showing bacterial exudate of the black chaff organism, Bacterium trans- 
lucens var. undulosum S., J. and R. X 15. 



24 



BACTERIAL DISEASES OF PLANTS 




Fig 16 -Black chaff of wheat. A gUune infection done by Lucia McCulloch 
in the summer of 1917 with No. 20, from McKinney, Texas. Inoculated June 21 
frota purTculture. Photographed July 9. The tiny beads are bactenal masses 
oozing from stomata. X 13. 



conspectus: agents of transmission 25 

Birds probably transmit some of these diseases on their 
feet or in other ways. In connection with the bud-rot of the 
coconut palm in the West Indies, I suspect the turkey-buzzard, 
but the evidence is not complete. Long since, Merton B. Waite 
obtained (once in Florida, once in Maryland) the strongest 
kind of circumstantial evidence going to show that pear blight 
may be spread by birds. 

Respecting insects, molluscs, and worms, the evidence is 
complete. They often serve to carry these diseases. I have 
summarized our knowledge in another place^ and will here 
content myself with a brief statement calling renewed atten- 
tion to the subject. 

We had very good evidence of the transmission of one bac- 
terial disease of plants (pear blight) by insects long before the 
animal pathologists awoke to the importance of the subject, ^ 
but it cannot be said that they have ever paid much attention 
to it, although it antedates by two years the work by Theobald 
Smith and Kilborne showing that Texas fever is transmitted by 
the cattle tick {Ixodes hovis Ry.) . That discovery also belongs to 
the credit of the United States Department of Agriculture, and the 
two together may be said to have laid broad and deep the foun- 
dations of this most important branch of modern pathology. 
Waite isolated the pear blight organism, grew it in pure cultures 
and proved its infectious nature by inoculations. With such 
proved cultures he sprayed clusters of pear flowers in places 
where the disease did not occur and obtained blossom-blight, 
and later saw this give rise to the bhght of the supporting branch, 
found the organism multiplying in the nectar, and re-isolated 
it from the blighting blossoms. On some trees he restricted the 
disease to the sprayed flowers by covering them with mosquito 
netting to keep away bees and other nectar-sipping insects. On 
other trees where the flowers were not covered he saw bees visit 
them, sip from the inoculated blossoms and afterwards visit 
blossoms on unsprayed parts of the tree, which then blighted. 

1 Smith, Erwin F.: "Bacteria in relation to plant diseases," Carnegie"^ Inst. 
Washington, Publ. 27, Vol. 2, p. 40, 1911. 

-Waite, Merton B.: Results from recent investigations in pear bhght, Bot. 
Gaz. 16, 259; Am. Assoc. Adv. Sci., Proc, 40, 315, 1891. 



26 



BACTERIAL DISEASES OF PLANTS 




I 



• 



# • 




// 



Fig 17 -Black chaff of wheat. Agar plate poured (June 2, 1917) from a leaf 
stripe of No. 20, McKinney, Texas. Photographed June 11. X 4. ^f^'^'^fl'- 
low and smooth on surface (as photographed), and also by direct transmitted 
light; but by obhque transmitted Hght they are like Figs. 18, 19. 



conspectus: agents of teansmksion 27 




Fig. 18. — Bacterium translucens var. uyidulosum Smith, Jones and Reddy, 
from black chaff of wheat. No. 662, Monticello, Illinois. Surface and buried 
yellow colonies on + 15 beef-peptone agar-poured plate. A pure-culture glume 
isolation of June 21, 1918. Photographed July 1 by oblique light to show internal 
markings — surface smooth. X 10. 



28 



BACTERIAL DISEASES OF PLANTS 




Pie 19 —Bacterium translucens var. undulosum S., J. R. from black chaff of 
wheat ■ No. 678, El Reno, Oklahoma. Surface and buried yellow colonies on 
+ 15 beef-peptone agar plate. Plated June 24, 1918 (from a glume). Photo- 
Japhed July 2, by oblique light; surface smooth. At x an mtrudmg colony. 

xio. 



conspectus: agents of transmission 



29 




Fig. 20. — Bacterium transluce'ns var. imdulosum S., J. R. from black chaff of 
wheat. No. 252, Republic, Missouri. Beef-peptone gelatin-poured plate (-|-9) 
from colony 52. Photographed by oblique light (from direction of the arrow) to 
show the small dry liquefaction pits. For the two at x, enlarged, see Fig. 21. 



30 BACTERIAL DISEASES OF PLANTS 

Finally he captured bees that had visited such infected blossoms, 
excised their mouth parts, and from these, on agar-poured 
plates, obtained Bacillus amylovorus, with colonies of which he 
again produced the disease. These experiments were done in 
several widely separated localities with identical results. I saw 
them and they made a great impression on me. 

The writer has since proved several diseases to be transmitted 
by insects, notably the wilt of cucurbits, and here the trans- 
mission is not purely accidental, but there appears to be an adap- 
tation, the striped beetle {Diabrolica vitiata) chiefly responsible 
for the spread of the disease being fonder of the diseased parts 
of the plant than of the healthy parts. This acquired taste, for 
it must be that, works great harm to melons, squashes, and cu- 
cumbers. Whether the organism winters over in the beetles, 
as I suspect, remains to be determined. Certainly the disease 
appears in bitten places on the leaves very soon after the spring 
advent of the beetles, i.e., before they have had opportunity 
to become infected from newly wilted cucurbits : 

In the summer of 1915, Mr. Frederick V. Rand, assistant pathologist in my 
laboratory, undertook, at my suggestion, two series of experiments on Long Island, 
N. Y., to determine the truth or error of this hypothesis. His results, which have 
afforded a striking confirmation of my views, may be summarized in brief as 
follows : 

In two cucumber fields where 75 per cent of the plants contracted the bacterial 
wilt disease in 1914 and where, up to September 1, 800 plants or about one in four 
contracted it in 1915 (later cases no doubt occurred but no further record was 
attempted owing to the appearance of the downy mildew), 180 plants kept inside 
of 50 large insect cages distributed at uniform distances through the two fields 
remained entirely free from the disease, except in two cages. In one of these two 
cages Diabrotica vittala was purposely introduced when the plants were only 2 to 
3 inches high, and before there was any of the disease on the check plants. In 
this cage all of the plants contracted the disease which first appeared in bitten 
places on the leaves. In the other cage, a single beetle of that species penetrated 
accidentally later in the season (when the disease was quite prevalent outside on 
the checks) and gnawed and infected a single plant before it was discovered and 
removed, the other, unbitten, plants in the cage remaining healthy. The beetles 
were collected in one of the two experimental fields, remote from other plantations, 
at a time when the check plants were small and all still free from the disease. It 
is believed, therefore, that they hibernated in the vicinity and that their last 
source of infection was diseased plants of the preceding year, i.e., that they carried 
the wilt organism over winter in their bodies. That not all hibernated beetles 
transmit the disease is shown by the fact that some were liberated at the same 
time in three other cages but the plants remained healthy, and by the additional 



conspectus: agents of transmission 



31 





Fig. 21.— Black chaff of wheat. Two small surface colonies on +9 beef pep- 
tone gelatin. Enlarged X85 to show thin pale fringe in the dry pit of liquefaction. 
Plate poured January 30, photographed February 9, 1918. 



32 



BACTERIAL DISEASES OF PLANTS 




Fig. 22. — Colonies of Bacterium iranducens var. undtdosum S., J. R., the cause of 
black chaff of wheat. Same as Fig. 21 but three days later. At the upper right 
side of the lower colony, lifted above the surface of the gelatin, is a bacterial tendril 
which has made 6 turns. X 85 



conspectus: agents of transmission 33 

fact that on the checks the disease first appeared on a few only of the many bitten 
plants, and from these few was subsequently spread to many others by the 
beetles, the disease appearing everywhere first, in bitten leaves, a few days after 
they were gnawed by the beetles. From these experiments we may conclude: 

1. Striking confirmation of my statements respecting summer distribution 
of this disease by Diabrotica viltata. 

2. Freedom of plants from disease when protected from this beetle by wire 
screens, although presumably growing in infected soil. 

3. Inability of aphides and flea beetles to cause the disease, since they entered 
the cages to some extent but did not act as carriers. 

4. Evidence that the disease is not air-borne or water-borne. 

5. Proof that the disease is not transmitted by way of the soil — at least not in 
the absence of insects. 

6. Strong circumstantial evidence that Bacillus tracheiphilus winters over in 
"bacillus carriers," i.e., in certain beetles which function as the spring distributors 
of the disease.' 

In 1897 I observed and proved experimentally that molluscs 
sometimes transmit brown rot of the cabbage, and in 1913 
I saw indications in Southern France which lead me to think that 
snails are responsible for the spread of the oleander tubercle, 
i.e., I saw them eating both sound and tubercular leaves, and 
found young tubercles developing in the eroded margins. 

Parasitic nematodes break the root-tissues and open the way 
for the entrance of Bacterium solanacearum into tobacco and 
tomato, as was first observed by Hunger in Java and later by 
myself in the United States. One of the serious problems of 
plant pathology is how to control the nematode, Heterodera radi- 
cicola, not only because of its wide distribution on a great variety 
of cultivated plants and the direct injury it works but also on 
account of the often very much greater injury it causes through 
the introduction into the roots of the plant of bacterial and fun- 
gous parasites. The man who shall discover an effective field 
remedy will deserve a monument more enduring than bronze. 
Parts of our Southern States in particular are overrun by this 
parasite. In the hothouse, of course, it may be controlled by 
steaming the soil, and in other ways. 

Much remains to be done before we shall know to what ex- 
tent fungous parasites function as carriers of parasitic bac- 
teria. H. Marshall Ward sought to explain the presence of 

'In most beetles, as shown by Rand's further studies (which will appear in 
Phytopathology) the ingested bacilli are promptly destroyed: in a few, they per- 
sist for a long time and are voided in the feces, which are then infectious. 



34 BACTERIAL DISEASES OF PLANTS 

bacteria in diseased plants by supposing that they must enter 
the plant through the lumen of fungous hyphae. In this he 
was wrong, certainly, if it be stated as a general proposition, 
since many bacteria are able to attack and do attack, unassisted, 
but it appears to be clear that in some cases the two types of 
parasites work together, the fungus invading first, and the bac- 
terium following hard after and often doing the major part of 
the damage, as in potatoes attacked by Phytophthora infestans. 
The reverse of this also occurs, the bacterium entering first and 
the fungus following, as in crown gall followed by Fusan'um. 
Parasitic bacteria are soon followed by saprophytic bacteria, 
which complete the destruction of the tissues, and, if the dis- 
ease is somewhat advanced, cultures from the tissues may 
yield only the latter (potato rots, lettuce rots, etc.)- Also, 
as in animals, one bacterial disease may follow another and the 
second be more destructive than the first, e.g., fire-blight on 
the apple following crown gall. 

EXTRA-VEGETAL HABITAT OF THE PARASITES 

Here is perhaps the place to say a few words about the non- 
parasitic life of the attacking bacteria. 

All are able to grow saprophytically, i.e., on culture media 
of one sort or another, and probably all live or may live for 
a time in the soil. Very few, however, have been cultivated 
from it. The vast mixture of organisms present in a good 
earth rather discourages search. In some of the unsuccessful 
attempts failure may have been due to having undertaken 
isolations at not exactly the right time, or in not just the right 
place, or on not just the proper medium, but more often prob- 
ably to the swamping tendency of rapidly growing saprophytes. 
How long a parasite is able to maintain its virulent life in a soil 
must depend largely on the kind of competitors it finds. I 
have used the term virulent, because it is conceivable that an 
organism might remain alive in a soil long after losing all 
power to infect plants, just as we know it can in culture media. 
Bacterium solanacearum causing brown rot of Solanaceae and of 
many other plants. Bacillus phytophthorus causing basal stem- 



conspectus: extra vegetal h.abitat 35 

rot and tuber-rot of the potato and Bacterium tiimefaciens, 
causing crown gall, sometimes certainly live in the soil, and the 
soundest plants when set in such soils, especially if wounded, 
are liable to contract the disease, if they belong to susceptible 
species. The root-nodule organism of Leguminosae, which I 
have not considered here, also lives in many soils, as every 
one knows. 

MORPHOLOGY AND CULTURAL CHARACTERS OF THE PARASITES 

]Most of the plant bacteria are small or medium sized rod- 
shaped organisms. They have rounded ends and are of variable 
length but are seldom more than 1^ in diameter and sometimes 
less than 0.5^- A ery few parasitic coccus forms are known; 
in fact, none are very well established, but animal diseases due 
to cocci occur and presumably there are such plant diseases. 
Some of these bacteria are Gram positive, others are not; few, 
if any, are acid fast. All takes stains, especially the basic 
anilin dyes, but not all stain with the same dj^e equally well. 
Alost of the species are motile b}- means of flagella — polar or 
peritrichiate; a few are non-motile, genus Aplanobacter.^ Some 
develop conspicuous capsules, others do not. Few, if any, pro- 
duce endospores. Under special conditions long filaments and 
chains are frequent. Under abnormal conditions many be- 
come club-shaped, y-shaped, or otherwise branched. Lohnis 
believes {Jour. Agr. Research, Vol. 6, July 31, 1916, p. 675) 
that all bacteria have an amorphous stage, but such is not my 
belief. Grown pure on culture media in mass, they are either 
yellow, pure white, or brownish or greenish from the liberation 
of soluble pigments. Red or purple plant parasites are not 
known. We formerly supposed that there were no green fluo- 
rescent species capable of parasitism, but now several are 
known: e.g., the organism causing the lilac blight of Holland 
[Bacterium syringae[C. J. J. van Hall] EFS), with pure cultures of 
which the writer was the first to obtain typical infections, at 
Amsterdam in 1906 (garden of the Willy Commelin Scholten 
Laboratory, courtesy of Johanna Westerdijk) and afterwards in 

1 Smith, Erwin F.: "Bacteria in relation to plant diseases." Carnegie Inst. 
Washington. Publ. 27, Vol. 1, p. 171, lOOo; Ihlil. 27. Vol. 3, pp. 1.55, 161, 1914. 



36 BACTERIAL DISEASES OF PLANTS 

the United States; Bacterium lachrymans^ the organism causing 
the angular leaf-spot of cucumber (Figs. 6 to 9 and 23 to 26); 
Bacterium aptatum Brown and Jamieson, causing leaf-spots on 
Tropaeolum and on beet; and some of the lettuce spot organisms 
{Bacterium viridilividu7n Brown, Bacterium marginale Brown). 

Some species produce gas (chiefly COo and H), liquefy 
gelatin, consume asparagin, destroy starch, and reduce ni- 
trates; others do not. Their fondness for sugars and alcohols 
is quite variable. Some are extremely sensitive to sunlight 
and dry air {Bacillus carotovorus, Bacillus tracheiphilus, 
Bacterium solanacearum, Bacterium malvacearum) ; others are 
remarkably resistant, remaining alive and infectious on dry 
seeds for a year {Bacterium campestre, Aplanohacter stewarti, 
Aplanobacter rathayi, Bacterium translucens) . Some are strictly 
aerobic, others can grow in the absence of air, if proper foods 
are available. Some are very sensitive to acids, alkalies and 
sodium chlorid, others are not. Some have wide ranges of 
growth from 0°C. upwards. Some will not grow at or near 
0°C., others will grow at or above 38°C. Very few, however, 
will grow at blood temperature, certain ones even in plants 
or on culture media are killed by hot summer temperatures, and 
none are known definitely to be animal parasites, unless we 
except Bacterium tumefaciens. My own animal experiments 
with this organism have been limited largely to efforts to produce 
tumors in fish and salamanders. Many of the trout died 
early of what appeared to be septicaemia and from the dorsal 
aorta of one of these fish the crown-gall organism was re-isolated 
in pure culture on agar poured plates and with subcultures 
from one of the colonics crown galls were induced on sugar beets. 
Other trout have yielded, both in the abdominal wall and in the 
eye-socket, what I regard as small tumors but no metastases 
have been observed. According to Friedmann, Bendix, Hassel 
and Magnus, Bacterium tumefaciens causes a purulent menin- 
gitis in man and also an ulceration of the intestinal mucosa 
{Zeits. f. Hygiene u. Infektionskr., April 23, 1915), but Jensen 
of Copenhagen has contradicted this, having shown that 
Friedmann's supposed pure culture was contaminated, and 
Friedmann himself now admits that he was in error. 



conspectus: morphology and cultural characters 37 




Fig. 23.— Angular leaf-spot of cucumber. Under-surface of an inoculated leaf 
showing the "tear-drop" exudate of Bacterium lachrymans Sm. and Br.y. Planar 
enlargement by James F. Brewer. Inoculated May 6, 1915. Photographed 
May 12. X 4 circa. 



38 



BACTERIAL DISEASES OF PLANTb 




Fig 24.— Buried and surface colonies of Bacterium Iqchrymans Sm. and Bry. 
on a rather thickly sown +10 peptone-beef-gelatin plate: the cause of angular 
leaf-spot of cucumber. Poured May 12, 1915. Photographed May 17. X 10. 



conspectus: morphology and cultural characters 39 




Fig. 25. — Surface and buried colonies of Bacterium lachrymans Sm. and Bry., 
on thin-sown plates: A. Peptone-beef gelatin (-hlO). Plate poured May 12, 
19L5. Photographed May 19 with oblique lighting to show the peculiar margin 
at a;. X 14. S. Peptone-beef agar (-f- 15). Photographed by direct transmitted 
light to show the internal structure of the smooth white surface colonies. Plate 
poured August 6, 1916. Photograplied August 9. X 14. 



40 



BACTERIAL DISEASES OF PLANTS 




PiQ 26 —A, Film of Bacterium lachnjmans floating on Cohn's solution and full 
of crystals. Inoculated November 23. Photographed November 28, 1914. X 13. 
B Bottom of an agar slant culture of Bacterium solanacearum from iropaeolum, 
showing crystals formed in the agar. Slant at S. Photographed September 23, 
1914. X 7. ^ 



conspectus: action on the plant 41 

action of the parasite on the plant 

In some cases it is hard to draw the Hne between parasitism 
and symbiosis or mutualism. Probably we shall find more 
and more of these transition states; undoubtedly there are many. 
I have included Ardisia in my list of genera and have excluded 
the genera of legumes subject only to root-nodules. But a 
nodule on the root of a legume, so far as the local condition is 



/*■ 




V-' 






tif. 



r 



Fig. 27. Fig. 28. 

Fig. 27. — Ardisia leaf showing swollen, white, bacterially invaded leaf-teeth- 
^^ nat. size. 

Fig. 28. — Bacterial cavity in leaf-tooth of Ardisia crispa. 

concerned, is a disease as much as a leaf-spot, and, if Nobbe 
and Hiltner's statements are to be credited the general effect 
of the root-nodule organism on the plant may be excessive and 
injurious and not to be distinguished from a disease.^ 

In the tropical East Indian Ardisia, which is one of the 
strangest cases of mutualism known to me, and on which Miehe 

1 Smith, Erwin F. : "Bacteria in relation to plant diseases," Carnegie Inst. 
Washington, Publ. 27, Vol. 2, 1911, p. 131, last paragraph. 



42 



BACTERIAL DISEASES OF PLANTS 



has done a beautiful piece of work, we have perhaps something 
akin to what occurs in the root-nodules of legumes. This is a 
common hothouse plant, grown for its ornamental red berries 
and thick evergreen foliage (Fig. 27). Here the bacterial injury 
is local and internal. The bacteria are most abundant in the 
leaf-teeth where they form pockets or cavities (Fig. 28) and multi- 




\%iiy^f.^-,rA^-'a^ 



Fig. 29. — Section of leaf- 
tooth of Ardisia, showing a water- 
pore {S) connecting with the bac- 
terial cavitv. 




Fig. 30. — A. B. Photographs of two leaves 
of Pavetta angustifolia from Java. One show- 
ing bacterial leaf knots; the other free. Re- 
duced. {Courtesy of Johanna Westerdijk.) 



ply enough to make the leaf-serratures appear blanched or yel- 
lowish and slightly swollen, but never enough to kill them, 
or cause the leaves to become yellow and fall. There are no 
superficial indications of disease, except that the leaf serratures 
gradually enlarge slightly, lose chlorophyll and become white. 
In smaller numbers the bacteria occur in other parts of the plant, 
including the inner parts of the seed from which they are trans- 



conspectus: action on the plant 



43 



mitted to the seedling, whose leaf-serratures, infected probably 
through their water-pores (Fig. 29), in turn become the chief 
focus^of the bacterial multiplication. Apparently the bacteria 
are always present, and we do not know what would happen to 











Fig. 31. Iig. 32. 

Fig. 31. — Cross-section of leaf of Pavetla angiistifolia showing a veinlet and a 
small bacterial nodule. The veinlet has a closed cylinder of x\'lem. The bacterial 
pockets in the young nodule are drawn in solid black. At c.c. are masses of collen- 
chyma. It looks as if the organisms entered from the upper surface very earty 
through the palisade tissue. Drawn November 21, 1914, from an unstained sec- 
tion lying in water. 

Fig. 32. — Structure of one of the leafknots shown in Fig. 30.4. Stained with 
anilin blue. 

Ardisia plants grown without them, nor do we know how to 
obtain such plants (1915). It would be an interesting experi- 
ment to see if they could be produced without the bacteria and 
to watch their behavior. 



44 



BACTERIAL DISEASES OF PLANTS 



Ardisia plants, so far as I have been able to observe, grow 
very slowly. Query: Are they dwarfed by the presence of the 
bacteria, or on the contrary if deprived of them would they be, 
unable to grow at all? I set one of my assistants at work on 
the problem, asking her to heat Ardisia seeds in water at tem- 
peratures between the killing point of the seeds and that of the 
bacteria (which is somewhat lower). The seeds heated for the 




Fig. 33. — Part of another leaf-nodule of Pavetta angustifolia (Fig. 30A) more 
highly magnified, showing many small cavities. 



right time at the proper temperature were not killed but germi- 
nated and grew, although with extreme slowness as compared 
with those growing from untreated seed, so that even after a 
year they had scarcely a leaf to show but only a swollen bud 
and some roots. Sections from the leaf-teeth of such plants 
showed them to be free from bacteria, and this makes it seem 
probable that Ardisia plants actually require the bacteria. A 



conspectus: action on the plant 45 

repetition of this experiment gave the same results — good growth 
of checks and astonishingly slow growth of plants from the 
heated seed. 

We are now (1918) growing Ardisia plants from surface- 
sterile seeds in flasks of glowed sand in nitrogen-free media. ' 

The bacterial nodules on the leaves of the East Indian 
Pavettas (first described by Zimmermann) are apparently of a 
similar nature, but here the bacterial foci are scattered over 
the surface of the leaf, often, however, with astonishing regu- 
larity (Figs. 30 to 34). 






•>' 



5< 



B-*-'- 



Fig. 34. — Bacteria from a nodule nu |i:ii of Pavetta angustifolia. X 1000. 
Stained witli anilin blue. 

The action of such organisms as I have just mentioned differs 
probably from the behavior of active parasites in that they 
liberate much weaker toxins and enzjaiies, can attack only very 
actively growing parts, and also give off compensating nitro- 
genous substances. Not yet proved for Ardisia (1915) but 
proved apparently for the Pavettas by Dr. F. C. von Faber whose 
paper is in Jahrb. f. wi/^s. Bot., 1914, Bd. 54, p. 243.2 

^ Dec, 1919. These plants have remained alive for IS months, but the foliage 
is paler and the plants have made less growth than checks in ordinarj' soil. The 
experiment is not conclusive because at the end Dr. Jodidi found a trace of 
nitrogen in the sand (0..5 nig. per kilo). 

- Since this was written Miehe claims to have proved it for Ardisia, but owing 
to the war I have not been able to obtain his paper, which is in Ber. d.d. Bot. Ges., 
xxxiv Bd., 1916, pp. 576-5S0. 



40 BACTERIAL DISEASES OF PLANTS 

The active parasites produce toxins freely, poisoning the 
tissues, and enzymes converting starches into sugars, com- 
plex sugars into simpler ones, and so on, for their nutrition. 
They also neutralize and consume plant acids, and feed upon 
amino bodies and other nitrogenous elements of the host. As 
a result of their growth, many of them liberate both acids and 
alkalis, to the detriment of the plant. The solvent action of 
their products on the pectin compounds of the middle lamellae 
separates cells and leads to the production of cavities in the 
cortex, pith, phloem and xylem. There is also, or may be, a 
mechanical splitting, tearing or crushing due to the enormous 
multiplication of the bacteria within confined spaces. The 
whole intercellular mechanism of soft plants may be honey- 
combed and flooded in this way, and if the cavities are near the 
surface the tissues may be lifted up or the bacteria may be forced 
to the surface through lenticels or stomata in the form of tiny 
beads or threads (pear, plum, bean, maize, sugar-cane, cotton, 
mulberry, etc.), or by a splitting process. The splitting in 
plum fruits and peach fruits, due to the black spot, results, 
however, from local death of the attacked tissue with continued 
growth of the surrounding uninjured parts. I now doul)t if 
any of these plant parasites consume true cellulose. 

A majority of the forms known to cause plant diseases are 
extra-cellular parasites occupying chiefly the vessels and inter- 
cellular spaces, causing vascular diseases, soft rots, spot dis- 
eases, etc.; but intra-cellular parasites also occur, e.g., Bac- 
terium leguminosanim^ causing root-nodules on legumes, and 
Bacterium tumefaciens causing crown gall. The former multi- 
plies within the cell myriadfold, prevents its division, destroys 
its contents including the nucleus, and enormously stretches 
the cell wall so that the cell becomes much larger than its normal 
fellow cells and is packed full of the bacteria. The latter 
does not multiply abundantly in the cell, does not enlarge it 
greatly, does not injure its viability, and would be a harmless 
messmate were it not for the fact that it exerts a stimulating 
effect on the nucleus, compelling the cell to divide again and again. 

I This is a polar fiagellato organism — usually it is 1-3 flagellate. 



conspectus: action on the plant 



47 








I 

imi 



Fig. 35. — .4. O'Gara's Aplnnobader agropyri on Agropijron smithii (the west- 
ern wheat grass). Utah, photographed from dried material collected bv P. J. 



48 BACTERIAL DISEASES OF PLANTS 

In 1911 from Maryland carnations^ and again in 1913 
from Danish orchard grass- the writer called attention to a 
new type of bacterial disease in which the principal growth of 
the parasite is on the surface of the plant, that is between closely 
appressed organs. Since then O'Gara has described a similar 
disease from wheat-grass in Utah^ and Hutchinson from wheat 
in India. ^ See Fig. 35. 

THE REACTION OF THE PLANT 

We now come to the reaction of the plant. What response 
does it make to this rude invasion? Twenty years ago we might 
have said, "With rare exceptions, the plant is passive or nearly 
so," but that would have been a superficial observation. In 
every disease we must suppose that the plant makes some effort 
to throw off the intruder, although often its forces are paralyzed 
and overcome very early in the progress of the disease. 

One of the most conspicuous results is lessened growth In 
some of my plants recovering from brown rot due to Bac- 
ierium solanacearum,^ a month after external signs of the 
disease had disappeared the check plants were twice the size 
of the inoculated ones, and there was still a very decided dif- 

1 Smith, Erwin F. : "Bacteria in relation to plant diseases," Carnegie Inst, of 
Washington, Publ. 27, Vol. 2, Fig. 4, Oct. 30, 1911. 

2 Smith, Erwin F. : A new type of bacterial disease. Science, n.s., Vol. 38, p. 
926, Dec. 26, 1913. For a fuller account with figures see " Bacteria in relation to 
plant diseases," Carnegie Inst, of Washington, No. 27, Vol. 3, pp. 15.5-160. 

^ O'Gara, P. J.: A bacterial disease of western wheat-grass, Agi'opyron Smilhii, 
Phytopathology, Vol. 6, No. 4, August, 1916, p. 341. 

^ Hutchinson, CM.: A bacterial disease of wheat in the Punjab. Memoirs ot 
the Department of Agriculture in India, Agr. Research Inst., Pusa, October, 1917, 
Bacteriological Series, Vol. 1, No. 7. 

^ Smith, Erwin F.: "Bacteria in relation to plant diseases," Carnegie Inst. Wash- 
ington, Publ. 27, Vol. 3, 1914, Plate 45-D. 

O'Gara in 1916. The slime dries brownish yellow and masses of it may be seen 
adhering to various parts of the spike; B. The same showing a knee -shaped culm 
bending; C, D. Hutchinson's wheat disease of the Punjab (India) said to be due 
to a polar flagellate schizomycete {Pseudomonas tritici Hutch.). All the spikelets 
are stuck together or overgrown with a mass of lemon yellow slime. In D there 
is bending of the culm. {After Hutchinson.) 



conspectus: eeaction of the plant 49 

ference after more than two months. See also Fig. 169 where 
the development of the potato shoot inoculated with Bacillus 
carotovorus has lagged behind its twin shoot. Even more 
striking retardation results were obtained by the writer and 
Mr. Godfrey (summer of 1918) on Ricinus cornrnunis and 
on Helianthus annuus, using the same organism (Figs. 127, 131). 
On potato plants attacked early by Bacterium solanacearum 
the tubers remain small. On maize attacked by Aplanohacter 
stewarti the ears are imperfect. Olive shoots inoculated and 
infected by Bacterium savastanoi are always dwarfed (Figs. 
299 and 300), and frequently the crown-gall dwarfings are very 
conspicuous. The dwarfings of melon and squash plants at- 
tacked by Bacillus tracheiphilus are also conspicuous. Unin- 
oculated sugar-cane stems soon surpass in height and vigor 
those successfully inoculated with Bacteriuin vascularum. I 
do not know how to explain this checked growth unless it be 
due to the paralyzing effect of absorbed toxins. 

Changes in color are also conspicuous. The attacked parts 
may become greener than normal, or fade to yellow, red, brown 
or black. In tomato fruits there is often a retarded ripening 
on the attacked side, with persistence of the chlorophyll. 
In certain leaf-spots also the leaf green persists in the vicinity 
of the spot while the rest of the leaf becomes yellow (bean- 
blight). Crown galls on daisy are greenish. On the contrary 
the teratoid parts of crown galls on tobacco and on cauliflower 
are often blanched. The male inflorescence of maize attacked 
by Aplanohacter stewarti ripens prematurely and becomes white 
(Fig. 101). 

Distortions of various kinds appear (leaves of bean, lilac, 
larkspur, hyacinth, mulberry, Persian walnut, etc.). The leaves 
of tomato plants attacked by Bacterium solanacearum are bent 
downward; so are the fronds of the coconut palm when at- 
tacked by the bacterial bud-rot (Fig. 4) . The leaves of potatoes 
attacked by Bacillus phytophthorus are sometimes bent upward 
and almost always the leaflets are rolled upward, from the 
edges. Knee-shaped- curvatures of the culms appear on 
Dactylis attacked by Aplanohacter rathayi, in the buds of the 
sugar-cane attacked by Cobb's disease, on Agropyron attacked 



50 BACTERIAL DISEASES OF PLANTS 

by O'Gara's disease (Fig. 35B), and on wheat attacked by 
Hutchinson's disease (Fig. 35Z)). 

Organs may be developed in excessive number or out of 
place, as roots or leafy shoots in crown gall, witch-brooms on 
Pinus, and incipient roots on the stems of tomato, tobacco, 
chrysanthemum, nasturtium, etc. Hunger found a bud on a 
tomato leaflet which he attributed to the stimulus of Bac- 
terium solanacearum but this may have been natural (see Fig. 
36) and an old paper by Duchartre^ who first discovered adventi- 
tious buds on the leaves of the tomato. 




Fig. 36. — Cross-section of middle part of a tomato leaf in the region of the 
midrib showing leafy shoots originating from the sulcus. Variety Livingston's 
Dwarf Aristocrat. I have rooted and grown these foliar shorfts into mature 
fruit-bearing plants. Photographed June 23, 1916. X 4. 

In various diseases the plant removes starch from the vicin- 
ity of the bacterial focus which it endeavors to wall off by the 
formation of a cork-barrier, and in this effort it is sometimes 
successful, if the parasite is growing slowly (Figs. 136, 174). In 
other cases (hyperplasias) starch is stored in the diseased parts. 

The most conspicuous response of the plant, however, is in 
the form of pathological overgrowths — cankers, tubercles, and 
tumors. Some of these are very striking, e.g., those on the ash, 
olive, citrus, beet {Bad. beticolum^), pine, oleander, and on a 
multitude of plants attacked by crown gall. In some of these 

1 Duchartre, P.: Sur des feuilles ramiferes de Tomates, Ann. d. Sci. Nat. 3 
Ser. Bot., Tome 19, Paris, 1853, pp. 241-251, Plate 14. 

^ See "Tuberculosis of Beets" in '"Crown Gall of Plants: Its Cause and 
Remedy." Bull. 213, U. S. Dept. Agr., Bu. PI. Ind., Washington, D. C, 1911, 
pp. 194-195, and plate XXXIV. 



conspectus: reaction of the plant 51 

growths, which are hyperplasias, there is a great multiplication 
and simplification of the parenchyma and a great reduction of 
the vascular system, but in crown galls produced in the torus 
of the sunflower (which is a very vascular tissue) there is an 
excessive number of vessels. There are also various other 
phenomena, chemical and physical, nearly related to what takes 
place in certain insect galls, that is, increase of sugars, starches, 
enzymes, acids; and structural simplifications and reversions 
to more primitive forms. What I mean by reversions may be 
seen by consulting my figures illustrating insect galls. In 
crown gall, cell-division under compulsion proceeds at such an 
abnormally rapid rate that the cells are forced to divide while 
still imm.ature, and in this way masses of small-celled, unripe 
(anaplastic) tissue arise (Figs. 353, 354) and these develop 
tumor-strands (Figs. 319 and 323 to 325) in which secondary 
tumors form — phenomena suggestive of what occurs in malig- 
nant animal tumors (Consult text of No. XIV and various 
plates and figures and, especially, read Jensen's recent (1918) 
Danish paper referred to under Literature of No, XIV). 

prevalence and geographical distribution 

Economically considered, bacterial diseases of plants may be 
classed as major or minor. Most of the leaf-spots would fall 
into the latter class. Various soft rots, blights and vascular 
diseases, being wide-spread and destructive to plants of great 
economic importance, may be classed as major diseases. 
Cankers and tumors would fall midway in such a grouping. 
Occasionally a minor disease, e.g., lettuce rot, celery rot,^ under 
conditions favorable to the parasite may assume great importance. 
This is especially true of leaf-diseases which attack the fruit, e.g., 
the black spot of plum and peach due to Bacterium pruni. the 
bean-blight due to Bacterium phaseoli, the angular leaf-spot of 
cotton due to Bacterium malvacearum, the African mango dis- 
ease due to Bacillus mangiferae, the black chaff disease of wheat 

^" The loss from this disease in the fiehl where 1 gathered the specimens was 
150 crates out of ever,y 700 crates packed." (Dr. J. Rosenbaum, Hastings, 
Fla. Letter of April 4, 191(3.) 



52 BACTERIAL DISEASES OF PLANTS 

due to Bacteriu7n translucens var. undulosum (see Figs. 12, 14, 
38) and citrus canker due to Bacterium citri. 

It will be of interest to mention a few of these diseases with 
particular reference to their distribution and prevalence. 

Dutch East Indies. — The tobacco disease of Sumatra and 
Java is probably the most destructive, if the Sereh of sugar- 
cane is not bacterial. Each of these diseases has caused enor- 
mous losses. Each threatens or has threatened an industry. The 
tobacco disease occurs also in the West Indies, in the United 
States, and probably also in South Africa. If Janse's root dis- 
ease of Erythrina, the coffee shade tree of Java, is also bacterial, 
as he supposed, then there is another great bacterial plague 
in that region, for hundreds of thousands of trees have died, 
and another species has been substituted as a shade tree. The 
brown bast disease of rubber trees {Hevea brasiliensis) , which 
is a tumor disease of the bast of suspected bacterial origin, is 
widespread and has attracted much attention in recent years. 
There is also a bacterial disease of peanuts. 

West Indies. — Here the most destructive disease is the bac- 
terial bud-rot of the coconut palm, which occurs all around the 
Caribbean, and threatens the entire destruction of a profitable 
industry in Cuba. There is also the bacterial disease of bananas 
and plantains, but the most wide-spread and destructive Musa 
disease of the Western Hemisphere is the Panama disease, due 
to Fusarium cubense EFS.'^ 

Australia. — Cobb's disease of sugar-cane has probably at- 
tracted more attention in Australia than any other bacterial 
trouble, although bacterial rots of the potato are also very 
destructive. The cane disease in both Queensland and New 
South Wales has in many cases destroyed the output of whole 
plantations and greatly discouraged planters. This disease 
occurs also in Fiji, and probably in South America. According 
to G. F. Hill, the citrus canker occurs in the Northern Territory 
of Australia (Bull. N. T., Austr., 18, 1918). 

1 On this subject see papers by Elmer W. Brandes as follows: (1) Ann. Rep., 
Porto Rico Agr. Exp. Sta. for 1916, pp. 29-31; (2) Distribution of Fusanuw 
cubense EFS, the cause of Banana wilt, 20th Report Mich. Acad, of Sciences, 
1918, pp. 271-275; and (3) Banana wilt. Phytopathology, Sept., 1919, pp. 339- 
389, 14 plates and 5 text figures. 



conspectus: prevalence and distribution 53 

Japan. — The tobacco wilt, which has destroyed many fields, 
is probably the worst Japanese disease. This is believed by Hon- 
ing and by the writer to be identical with the tobacco wilt of 
Sumatra and of the United States. The citrus canker occurs 
and several other interesting bacterial blights have been re- 
ported from Japan, including one on the basket willow (Fig. 37). 




Fig. 37. — Agar poured plate colony of the schizomycete causing the Jajjanese 
basket-willow disease. Photographed by oblique transmitted light to show inter- 
nal structure. X 10. 

China. — In the interior of China there is a destructive wilt 
disease of tobacco (Frank N. Meyer), but its nature is unknown. 
A Fusarium cultivated from it in my laboratory did not cause 
the wilt when I inoculated it copiously into the soil near broken 
roots of young tobaccos nor yet when I introduced it into deep 
wounds made in the stems of young vigorous plants at the sur- 
face of the earth. 



54 BACTERIAL DISEASES OF PLANTS 

The Philippines. — In Luzon, citrus canker, a bud-rot of coco- 
nut, brown rot of potatoes and egg plants, a leaf spot of tobacco, 
bean blight, a bacterial rot of bananas and Musa textilis, and some 
other diseases occur. Most of the islands are pathologically 
unexplored. According to Reinking (The Philippine Journal 
of Science, Vol. XIV, Jan., 1919, pp. 131-151) the coconut bud- 
rot of the Philippines is due to Phytophthor a faheri Maubl., Bacil- 
lus coli and a schizomycete resembling B. coli isolated from the 
rotting palm bud may aggravate the rot but cannot initiate 
it except under very favorable conditions of moisture and pre- 
vious injury. 

India. — The brown rot of Solanacece is common and destruc- 
tive. Citrus canker is common especially in the Punjab (Hutch- 
inson, in a letter to the writer). There is also a bacterial disease 
of the opium poppy. Most of Asia is a terra incognita. 

South Africa. — The mango disease in recent years has greatly 
reduced the exports. Potato and tomato wilts are common. 
There is a serious tobacco disease, probably bacterial. Crown 
gall is common and injurious on shade and orchard trees. An- 
gular leaf-spot of cotton is prevalent. Other bacterial diseases 
occur, including several on citrus. Nothing is known about the 
greater part of Africa. 

South America. — There is a serious disease of sugar-cane 
in Brazil and another in Argentina, both of which I believe are 
of bacterial origin and identical with Cobb's disease, but this 
remains to be proved. Bondar has reported a destructive mani- 
hot disease. The bud-rot of the coconut occurs in the north. 
The banana disease of Guiana, however, is due to Fusarium 
cuhense. Most of South America, like Asia, is unexplored. 

United States and Canada. — Potato rots of which we have 
several distinct forms, probably cause the greatest losses, one 
year with another. Following these I should think pear and 
apple blight. Perhaps the latter should be placed first, for the 
destruction of an acre of potatoes would scarcely equal the value 
of a single fine pear tree, and thousands are destroyed every 
year. In Cahfornia, which was formerly free from pear bhght, 
the losses in the last twenty years have been enormous, amounting 
to about one-third of all the full-grown orchards and to a money- 



conspectus: prevai^ence and distribution 55 

loss estimated at $10,000,000 for the five years preceding the 
efforts for its restriction begun in 1905 by the United States 
Department of Agriculture. This is a very conservative esti- 
mate considering the number of trees destroyed. In the San 
Joaquin Valley in California, "in the short space of three years, 
from 1900 to 1904," according to O'Gara, "almost half a million 
pear trees were lost by blight. Practically no attempt was made 
to check the disease and one of the greatest industries of the 
San Joaquin Valley vanished like a dream." Very serious 
losses from this disease are experienced every year in the East, 
or were until growers became generally familiar with methods of 
control. In certain seasons bacterial diseases of barley and oats 
injure these crops to a considerable extent. 

In our southern states the wilt disease of tobacco and the 
tomato, due to Bacterium solanacearutn , has made it impossible 
to grow these crops on many fields. In the northern United 
States the cucurbit wilt is wide-spread and destructive, but 
cucurbits are, of course, a minor crop. Blight of beans due 
to Bacterium phaseoli is another common and troublesome dis- 
ease. In certain seasons and on some varieties the angular 
leaf-spot injures cotton very seriously (see Part III, No. X). 

The wide prevalence and destructive nature of the bacterial 
black chaff of wheat in the United States west of the Mississippi 
River in 1915, and since, adds another to our serious bacterial 
diseases. This blights the leaves, shortens the head and shriv- 
els the kernels (Fig. 38). In the study of this disease in my labo- 
ratory during the last three years we have discovered a second 
bacterial disease of wheat previously confused with the ''black 
chaff," the basal glume rot (Figs. 39, 40), due to Bacterium 
atrofaciens McCuUoch, a green fluorescent organism which causes 
a black rot at the base of the kernel. 

The walnut blight has done much damage in California and 
recently it has been reported from New Jersey (Cook) and from 
other parts of the Eastern United States (McMurran, Bull. 
611, U. S. Dept., Agric). This disease occurs also in ChiU, 
South Africa (Miss Doidge: Letter to the Author), New Zealand 
and Tasmania. 

The bacterial disease of alfalfa has been serious in parts 



56 



BACTERIAL DISEASES OF PLANTS 




Fig. 38— Kernels of Russian winter wheat attacked and shriveled by the 
black chaff organism, Bacterium iranslucensvar. undulosum, S., J. R., Coll. No. 271 A, 
Kansas, 1917. All kernels from one head and all but 8 shriveled. Bacterial hlms 
can be seen on the shriveled kernels, especially those of the middle row. 



conspectus: prevalence and distribution 



57 




Fig. 39. — Head of wheat, from Kansas crop of 1917, Coll. Xo. 47S, .showing 
basal glume rot, a new disease, due to Bacterium atrofaciens McCuUoch, a white 
organism causing a green fluorescence in media. 



58 



BACTERIAL DISEASES OF PLANTS 




Fig. 40. — Glumes and kernels of wheat blackened by Bacterium atrofaciens 
McCuUoch, the cause of the basal glume rot, crop of 1917, Coll. No. 285 (New- 
York) and No. 399 (Canada). 



conspectus: prevalence and distribution 



59 



4 





ft 



i?^. 
^'^-.-M 



A 













Fig. 41. — Bacterial canker on leaves of grape-fruit from Florida: (1) A natural 
infection, 1914; (2) an inoculation of 1914. Both leaves were deposited in the 
Pathological Collections, B. P. I., U. S. Department of Agriculture, in June, 1914, 
by H. E. Stevens of Florida. No. 2, designated as a " pure-culture inocula- 
tion," was supposed by Stevens to have been caused by his fungus (Phoma or 
Phyllocticta) which is present, but is not the parasite. 



60 



BACTERIAL DISEASES OF PLANTS 





Fig. 42.— Citrus canker on leaf of seedling grape-fruit. Inoculated by the 
writer with Bacterium citri (Hasse) Jehle, isolated from a grape-fruit leaf received 
from Mississippi in 1915. Time, 16 days. Cankers not \et ruptured X 5 



conspectus: prevalence and distribution 



61 



of the West, but I have not heard of its occurrence in the East. 
It is most injurious early in the season, i.e., on the first cutting. 
Alfalfa is now, according to Piper^ the third most important 
forage crop in the United States, only timothy and red clover 
exceeding it. There is a physiological "white spot" on alfalfa 
(O'Gara) not to be confused with Sackett's disease. 






Fig. 43. — Citrus canker due to Bacterium citri (Hasse) Jehle: A, on Citrus 
decumana (grape fruit) ; B, on Citrus trifoliata. Disease introduced into America 
recently from Eastern Asia. 



Recently in Florida the citrus canker (see Figs. 41 to 44) 
has caused orange growers a great scare and strenuous efforts 
are on foot to suppress it. During the last four years, under 
pressure from the citrous States, the General Government of the 
United States has made five appropriations for this purpose 

1 Piper, Chas. V.: Forage plants and their culture, N. Y., The Macmillan Co., 
1914. 



62 



BACTERIAL DISEASES OF PLANTS 



as follows: Special, January, 1915, $35,000; special, February, 
1916, $300,000; general, in Department of Agriculture appro- 
priation billy, 1916-17, $250,000; 1917-18, $430,000; 1918- 
1919, $250,000.' This disease occurs also in Japan, China and 
the Philippines (Water T. Swingle) and was certainly intro- 
duced into the United States on imported citrus plants. It 

now occurs (or did occur in 1916) 
in every Gulf State. It should not 
be confused with the somewhat similar 
looking Costa Rican pseudo-canker 
(Figs. 45, 46) which is of non-bacterial 
origin, nor with the verrucosities due 
to Cladosporium citri (Fig. 47). The 
true bacterial canker is usually sur- 
rounded by a narrow water-soaked 
area best seen by holding the leaf up 
to the light (Fig. 48) and is swarm- 
ing with bacteria, whereas the pseudo- 
canker shows no such border and con- 
tains at most only some fungous 
threads well corked out. 

Holland and Denmark. — In Hol- 
land the yellow disease of hyacinths 
will eventually put an end to hyacinth- 
growing for export if means cannot be 
had for its control, since the land 
suited to hyacinths is limited in 
amount. Black rot of cabbage oc- 
curs in Holland and Denmark, and is common now also in 
many parts of the United States. It was probably imported 
into the United States from Denmark on cabbage seed. 
Some years in nurseries about Amsterdam the lilac blight has 
been troublesome. In Denmark Rathay's disease is said to be 
rather troublesome on orchard grass grown for seed. 

1 On April 30, 1918, in the Florida Plant Commissioner's Office, Department 
of Citrus Canker Eradication, 183 persons were employed, including a divisional 
inspector, district inspectors, assistant district inspectors, foremen and inspectors. 
During the year 1918 over 2,000,000 grove trees were inspected and six times as 
manj^ nursery trees. 




Fig. 44. — Cross-.section of 
grape-fruit leaf showing a 
young canker inoculated by 
the writer in 1915. Time, 16 
days. See Fig. 42. 



conspectus: prevalence and distribution 



63 



M^^ 



-•K^ 



/I 




Pig. 45. — Pseudo-canker on Citrus aurantium, probably scab due to cladn- 
sporium citri. Some mycelium is present but no spores. The cankers have 
healed, being cut off from the rest of the leaf by a cork layer. Costa Rica, 1913. 



64 



BACTERIAL DISEASES OF PLANTS 



Sandwich Islands. — There is a serious banana disease but its 
cause is not known (1915). It attacks the Chinese banana. 
There is a serious potato disease and a bad shade tree disease, 
both of unknown origin. The mosaic of sugar-cane occurs. 

Great Britain and Germany. — Until recently in these countries 
not much critical study was given to bacteria as a cause of plant 




Fig. 46. — (Josta Kicau peudu-canker of citrus. A detail from Fig. 45, further 
enlarged to show absence of any translucent border such as that shown in Fig. 
48. Photographed by transmitted light. X 6. 

diseases, but now good students are at work. Potato rots are 
probably the most destructive bacterial diseases. Appel has 
described one and Spieckermann another. The bacterial 
potato rot Pethybridge and Murphy described from Ireland^ is 
like the German ''black leg." Wormald has described a rot 
of celery which is common also in the United States (Fig. 49). 

1 Proc. R. Irish Acad., vol. xxix, Sect. B, No. 1, 1911. 



conspectus: prevalence and distribution 



65 




Fig. 47. — Verrucosities on an orange leaf from Florida, due to the fungus 
Cladosporiuin cif.ri. Photo by Brewer, but on a Seed's plate which shows no dis- 
tinction between the pale green of the leaf and the dull yellow of the scabs. See 
page 121. 
5 



66 BACTERIAL DISEASES OF PLANTS 

Potter has written on a rot of swedes and Paine on a rot of 
mushrooms and a leaf-spot of Protea. 

France and Italy. — Potato diseases are common and at times 
very destructive. OUve tubercle, common also in California, 
and all around the Mediterranean, is prevalent in spots. Vine 
diseases, especially Maladie d'Oleran and crown gall, do con- 
siderable damage. Pear blight seems to be absent in France, 
but has been reported from several places in Italy. Mul- 
berry blight occurs. The destructive Italian rice disease, 




Fig. 48. — Bacterial citrus canker enlarged and photographed by transmitted light 
to show translucent border. X 6. The tiny white specks are oil glands. 

brusone, is not due to bacteria as reported, but to a fungus 
{Piricularia) . Not much exact work has been done on bacterial 
diseases of plants either in France or Italy. 

Spain and Portugal. — These countries are a terra incognita. 

Russia. — A few years previous to the late war there was a 
great awakening in Russia. A plant pathological journal was 
founded and numerous discoveries were reported. From this j our- 
nal and other sources it is evident that many bacterial diseases 
of plants occur. I think, for example, that our black chafT of 
wheat is an importation from Russia. At least it should be 



conspectus: prevalence and distribution 67 




Fig. 49.— Celery plants 24 hours after needle pricks introducing Wormald's 
celery-rot organism {Bacillus apiovorus). Photographed July 22, 1914. Natural 
size. 



68 BACTEEIAL DISEASES OF PLANTS 

searched for in that country. It was not observed in the wheat 
region West of the Mississippi River until after numerous im- 
portations of Russian wheats. 

METHODS OF CONTROL 

In conclusion, some words on prophylaxis will be in order. 
Until recently almost nothing was known. Unfortunately so 
far as regards most of these diseases, methods of control must 
still be worked out. But with rapidly increasing knowledge 
of the biological pecuharities of the parasites causing these 
diseases, and of the ways in which they are disseminated, light 
begins to dawn, so that before many years have passed we 
may confidently expect the more intelligent part of the public 
to be applying sound rules for the control of these diseases — 
rules based on the individual peculiarities of the parasites and 
carefully worked out experimentally by the plant pathologist. 
In the United States within a generation every large crop 
establishment will have its plant pathologist. The little 
that we now know may be summarized as follows : 

Waite has shown that pear blight winters over in excep- 
tional trees on trunk and limbs in the form of patches which 
ooze living bacteria the following spring (see Figs. 282, 283) 
and are visited by bees and other insects, and that if these 
''hold-over" spots are cut out thoroughly over regions several 
miles in diameter (wide as a bee flies), the disease does not ap- 
pear on the blossoms and shoots the following spring, except 
as it is introduced into the margins of this area from remoter 
uncontrolled districts. He has tried this method of control very 
successfully, both in Georgia and California. Sometimes only 
one tree in many carries over the disease, but such is not always 
the case, as Sackett has shown, and the success of this method 
involves the inspection of every pome tree in a district, with 
•complete eradication of every case of the hold-over blight, and 
this in great fruit regions requires a small army of trained 
inspectors. During the blighting period in late spring and 
early summer, if one would save his orchard, the trees must be 
cut over for removal of diseased material as often as every 
week, and in the worst weather oftener. Furthermore, some 



conspectus: methods of control 6^ 

of the stone fruits, e.g., apricots and plums, and various wild 
plants of the Family Rosaceoe, must be inspected because these 
also are subject to the blight. 

The introduction of diseases transmitted by way of seeds, 
bulbs, and tubers may be avoided by obtaining these from 
plants not subject to the disease. As this freedom cannot 
always be known, bulbs and tubers shovild be inspected criti- 
cally before planting, and firm-coated seeds should be soaked 
for 15 minutes in 1:1000 mercuric chlorid water; thin coated 
seeds susceptible to mercuric chlorid, e.g., wheat, in 1:1000 
copper sulphate solution (20 minutes) followed for a moment 
by milk of lime; or exposed to formaldehyd (40 per cent, for- 
malin 1 part, water 400 parts, for 10 minutes and then held moist 
for some hours). In case of five plants (cabbage, maize, wheat, 
barley and oats) we know positively that the diseases are trans- 
mitted on the seed and this is probably true for several others — - 
peas, beans, soybeans, cucumber (angular leaf spot^), sorghum, 
orchard grass. All shrivelled seeds should be screened out before 
planting. Dry beans will endure at least 10 minutes exposure 
to 1:1000 mercuric chlorid water and germinate freely. I have 
not tried longer exposures. The germination of wheat is in- 
jured even by 10 minutes exposure to 1:1000 mercuric chlorid 
water and very seriously by i^oo formalin water, and also 
by heavy doses of copper sulphate such as have been recom- 
mended for the elimination of smuts. A. G. Johnson has re- 
cently recommended hot dry air for the destruction of fungous 
and bacterial parasites on various grains, but tried on wheat 
for black chaff I found that either it seriously injured germina- 
tion or failed to destroy all of the bacteria. It may serve ad- 
mirably, however, for plot experiments where the aim is simply 
to procure sound grain for subsequent field sowings, since here 
a loss even of one-third of the seed grain is of no consequence 
in comparison with the end in view. Formaldehyd (formalin), 
if properly used, destroys all of the black chaff organisms 
on the surface of wheat kernels (Fig. 50) but kills some of the 

i"I saw remarkably virulent development of Bacterium lachrymaiis this 
summer [in Wisconsin] introduced on seed and spreading rapidly" (L. R. Jones, 
1919: Letter to the author). 



70 



BACTERIAL DISEASES OF PLANTS 




-piG 50. — Bacterium translucens var. undulosum, the cause of black chaff of 
wheat (Isolation No. 286, Island Park, Iowa) showing killing effect of 10-minute 
exposure to i^oo formalin water: (See next page.) 



conspectus: methods of control 71 

kernels and retards germination of the rest. The whole sub- 
ject of germicidal treatment needs careful revision. 

Since the above was written Harrj^ Braun of my laboratory 
has discovered (1919) that injury to seed-wheat, which is 
considerable when formalin solutions or copper sulphate solu- 
tions are used, is eliminated by soaking the seed in water for 
10 minutes and then keeping it moist for 6 hours before 
treating it. This allows it to absorb sufficient water to become 
resistant, since most of the injury is caused by the for- 
maldehyd that is carried into the grain along with the imbibition 
water. It is then soaked in one part of formalin to 400 parts 
of water for 10 minutes, drained, and covered for six hours to get 
effective action of the remaining aldehyd vapor on all the 
parasites, whereupon it is dried and planted. His work, re- 
peated many times on different varieties of wheat and to some 
extent also on other grains, shows nearly as full germination 
and quite as good growth in the treated plots as in the control 
plots. In fact, growth from the treated seeds is stimulated a 
little (Fig. 51). 

In studying pear blight Reimer found (1918) that 1 part 
of formalin in 9 parts of water is very effective for destroying 
the Bacillus amylovorus both on tools and in the tree wounds. 
He also found 3^:500 cyanide of mercury in water destroyed the 
pear blight organism in tree wounds effectively (with some slight 
injury to the wound) while Bordeaux paste or I500 mercuric 
chlorid water often failed. His ^ iooo cyanid of mercury 
water was not always effective, and he still has formalin treat- 
ment and the proper dose of cyanid under consideration 
(see Part III, No. XII). The cyanides, it should be remem- 
bered, are deadly poisons to man and the domestic animals. 



A. Check. Each kernel is surrounded by a pure culture of the yellow slime. 

B. Treated seeds. All are free from bacteria. The seeds were first baked to 
kill surface saprophytes, then soaked in a thick bouillon suspension of the black- 
chaff organism made from a young agar culture. Dried a day or two, exposed to 
the formalin and then planted on the nutrient agar. 

Nine active isolations of the black-chaff organism were tested in this seiies. 
Of the 894 formalin treated seeds only 13 showed any growth of the black-chaff 
organism after 8 days on the mitrient agar. Of the .528 check seeds all but 5 
developed colonies of the black-chaff bacterium. Photographed December 4, 1918. 



72 



BACTERIAL DISEASES OF PLANTS 




conspectus: methods of control 73 

The seed bed in case of tobacco, tomato, cabbage, and trans- 
planted plants generally, should be made on steam-heated or 
fire-heated soil, or on new earth which one has good reason to 
think free from the parasite in question. 

In the greenhouse, nematode-infested soils should be avoided 
or steamed or drenched with formalin water (one pint of forma- 
lin to 40 gallons of water). 

Cuttings of carnations, chrysanthemums, roses, peaches, 
plums, apples, quinces, sugar-cane, etc., used for slips, buds, 
or grafts should be from sound plants. By following this 
practice, recommended by Cobb, the more intelligent sugar-cane 
planters in New South Wales, it is said, have overcome the 
disease due to Bacterium vascularum. 

Commercial growers of hothouse roses in Maryland, New 
Jersey, New England and Canada have been much troubled 
in recent years by crown gall. To avoid this disease in houses 
great care should be exercised in the selection of soil and of 
cuttings. A knife used on diseased roses must not be used again 
on sound plants until disinfected. When a soil has become in- 
fected it may be steamed under weighted sheet-iron pans for an 
hour at 65 pounds pressure, by use of the autoclave (Fig. 56), 
by the steam-chamber (Fig. 57), or by means of a steam-drag 



Fig. 51.^ — Braun's imbibition experiments with seed wheat to prevent injury 
when subsequent!}^ treated with germicides. Formalin in distilled water (34oo) 
was used but the same results have been obtained with copper sulphate in water 
(1:80) and with formalin water (3-32o)- In each pot 100 seeds were used: 

VIII. Fulcaster wheat, planted March 8, 1918: photographed March 14. 

A. Soaked in formalin-water for 10 minutes (the usual method), then covered 
for 6 hours to get penetrating effect of the formalin vapor. Pot shows much re- 
tardation and killing. Germination 68 per cent. 

B. Check. Germination 85 per cent. 

C. Seeds presoaked, then treated with formalin as in A. By "presoaking" is 
meant that the seeds were plunged into water for 10 minutes, then removed 
and covered for 6 hours to keep moist. Germination 81 per cent. Plants show 
stimulation. 

X. Poole wheat, planted March 20, 1919; photographed March 26. 

A. Seeds presoaked (10 minutes in water) then covered 6 hours to keep moist. 
Treated 10 minutes in formalin-water, removed and covered for 6 hours. Then 
planted. Pot shows stimulation and 86 per cent, germination. 

B. Check; 95 per cent, germination. 

C. Duplicate of A. Shows stimulation and 89 per cent, germination. 



74 BACTERIAL DISEASES OF PLANTS 

which resembles a land-drag but is made of steam pipes, the 
teeth as well as the frame- work being hollow. The teeth are 
pointed and perforated with small holes so that when they are 
driven into the ground and live steam turned on it escapes and 
permeates the entire soil. I have seen the soil of lettuce houses 
in New England freed from parasites in this way. (Made by 
Geo. M. D. Sargent, Belmont, Mass.) 

On badly infested fields, whatever the disease, a careful 
rotation should be practised and low places should be drained. 

Certain diseases may be held in check by germicidal sprays. 
Newton B. Pierce reduced the number of infections in walnut 
blight in California 50 per cent by this method, using Bordeaux 
mixture. Scott and Rorer combated leaf-spot of the peach 
in this w^ay using self-boiled lime sulphur, the sprayed trees 
retaining their leaves, the unsprayed ones becoming defoliated. 
G. Bellini in Italy has recommended Bordeaux mixture and 
used it successfully on olive trees, following hail-storms, to 
keep out the olive tubercle. 

When, as in case of the cucurbit wilt due to Bacillus trach- 
eiphilus, diseases are transmitted by insects, destruction of the 
latter must receive prompt attention. Trap crops may be 
used. 

Great care should be taken to keep the manure heap free 
from infection. Diseased rubbish should be burned or buried 
deeply. It must not be thrown into a water-supply or fed to 
stock or dumped into the barnyard. Diseased potatoes and 
other crops should be cooked before feeding to animals. 

One of my fancies is that plant pathologists will eventually 
discover competing saprophytes which when sown on infected 
soils will overcome and render harmless certain of the bacterial 
parasites present in them. We have some evidence that nature 
does this, and man working toward a definite end should be able 
to improve on nature. 

Wet fields should be drained and, in general, every effort 
should be made to put the crop under good growing conditions, 
i.e., to give it a proper soil, good food, the right climate and 
the right amount of soil moisture. A wise rotation should 
also be practised. Rotation is the keynote of successful agri- 



conspectus: methods of control 75 

culture. Thefman who grows one crop year after year on the 
same soil|invites disaster. 

It has jeen found that some cultivated varieties are less sub- 
ject to disease than others (pear, apple, plum, rose, maize, beans, 
soy beans, potato, tomato, sugar-cane, banana, cabbage, etc.) , and 
there are also individual variations within the variety. These 
phenomena lead us to hope that by selection, or hybridization, 
valuable resistant strains may be originated. Meanwhile the 
resistant sorts when they are of any value commercially 
should be substituted for sensitive sorts in localities much 
subject to the disease. Unfortunately some of the resistant 
sorts have other less desirable qualities. A vast amount of 
experimental work must be done in this field before we shall 
have substantial results, and at least a generation or two will 
be required to learn even the boundaries of the field. But the 
problem offered is so enticing, and has such immediately practi- 
cal bearings on the food-supply of the w^orld, that in the near 
future we may suppose many pathologists will devote themselves 
to it, and that long before the whole field is worked over, many 
useful results will be forthcoming. The labor involved is enor- 
mous and exacting to discouragement at times, the results 
come so slowly, so much must be done to be certain of so little, 
all because the organisms dealt with are very small — hoiv small, 
we seldom realize! If the inhabitants of the United States or of 
Great Britain were reduced to the size of the smaller bacteria 
the entire population could occupy the surface of a silver dollar 
or of an English penny — and that too without crowding! 
O'Gara's happy characterization of the Fire-blight organism, 
"under the microscope, when magnified 1,000 diameters, its 
appearance is that of a hyphen '-' " applies equally well to 
nearly or quite all of the forms described in this book. 



PART II 
METHODS OF RESEARCH 

A few pages on the technics of plant bacteriology will be 
of service to the student. These are not designed to do away 
with the need of reference books. Therefore, at the beginning, 
the student is advised to read as much as he has time in the fol- 
lowing standard works, and to consult them daily as problems 
arise. The list is by no means exhaustive and does not include 
all the good books, but is more than sufficient probably for the 
beginner. 

A FEW OF THE MORE RECENT REFERENCE BOOKS 

Lee: The Microtomists' Vade-mecum. 7th ed. Phila- 
delphia, P. Blakiston's Son & Co., 1913. 

Gage: The Microscope. 12th ed. Comstock Pub. Co., 
Ithaca, N. Y., 1917. 

Mallory and Wright: Pathological Technique, W. B. 
Saunders Company, Philadelphia and London. 

Jordan: A Text Book of General Bacteriology. W. B. 
Saunders Company, Philadelphia and London. 

Eyre: Bacteriological Technique. W. B. Saunders Com- 
pany, Philadelphia and London. 

MuiR and Ritchie: Manual of Bacteriology. The Alac- 
millan Co., New York and London. 

Hewlett: Manual of Bacteriology. Clinical and Applied. 
5th ed. J. and A. Churchill, London, 1914. 

Stitt: Practical Bacteriology, Blood Work and Animal 
Parasitology, including Bacteriological Keys, Zoological Tables 
and Explanatory Clinical Notes. 5th ed. P. Blakiston's 
Son & Co., Philadelphia, 1918. 

Abbott: The Principles of Bacteriology. A practical 
manual for students and physicians. 9th ed. Lea and Febiger, 
Philadelphia and New York, 1915. 

76 



METHODS OF RESEARCH: REFERENCE BOOKS 77 

Park, Williams and Krumwiede: Pathogenic Micro- 
organisms. A practical manual for students, physicians and 
health officers. 6th ed. Lea and Febiger, New York and 
Philadelphia, 1917. 

Kendall: Bacteriology. General, Pathological and In- 
testinal. Lea and Febiger, Philadelphia and New York. 

Microbiology: A text-book of microorganisms general and 
applied. By Charles E. Marshall and many others. Second 
edition revised and enlarged with 186 illustrations. Published 
by P. Blakiston's Son & Co., Philadelphia, 1917. 

Laboratory Methods of the United States Army. Compiled 
by the Division of Infectious Diseases and Laboratories, Office 
of the Surgeon-General, War Department, Washington, D. C. 
Medical War Manual No. 6, pp. 256. Lea and Febiger, Philadel- 
phia and New York. A marvel of compactness and accuracy. 
It weighs only 4 ounces and is a veritable Vade-mecum. 

RiDGWAY, Robert: Color Standards and Color Nomen- 
clature. Washington, D. C, 1912. Pubhshed by the author. 
I have referred to this book as "R2" and to its predecessor, 
''A Nomenclature of Colors for Naturalists" (Little, Brown and 
Co., Boston, 1886), as "Ri." 

APPARATUS 

Only some hints which may prove useful are here included. 

1. For Preparation of Culture Media 

Culture media can be made, in default of better appliances, 
with some neutral litmus paper, a glass graduate, a pair of scales, 
some cork-stoppered flasks or bottles, a pen-knife, a kitchen 
stove and a tea-kettle, but certain other and more convenient 
kinds of apparatus are desirable. I shall mention only some of 
the leading articles. 

Receptacles. — Test tubes, beakers, pipettes, graduates, 
Erlenmeyer flasks, and Petri dishes are the most commonly used 
glassware. Resistant (insoluble) Jena glass or its equivalent, 
American Pyrex glass, is always desirable and is absolutely 
necessary for some purposes. 



78 BACTERIAL DISEASES OF PLANTS 

Cotton and Gauze. — The best surgeon's roll-cotton is not 
too good for plugging test tubes and flasks. Surgeon's gauze in 
bolts should be on hand for coarse filtering and various other 
uses. 

Balances. — There must be coarse pan-balances for ordinary- 
weighings, and fine balances for the more delicate operations, 
these latter usually under lock and key, especially where careless 
persons are wandering about. The Becker chemical balance, 
and the Kny-Scheerer analytical balance (Sartorius model) are 
very good. 

Dry Ovens. — The best dry oven I know is Lautenschlager's, 
This gives in all parts a very uniform temperature, and requires 
a minimum of watching. 

Steamers. — The Boston Board of Health steamer, made by 
the Arnold Steam Sterilizer Company, Rochester, New York, 
is recommended. 

Autoclaves. — I use two kinds: an upright autoclave made in 
Paris by P. Lequex, known as the Chamberland-Wiesnegg 
(depth 17 inches, diameter 13 inches), and a larger horizontal 
apparatus made by the Kny-Scheerer Company, New York, 
N. Y. The latter has a capacity of twelve 2-liter flasks, the 
depth being 28 inches, and the inside diameter 20 inches. By 
adjusting a valve, air is pumped out of the inner chamber and 
by another turn steam is allowed to enter whereupon the two 
pressure gauges should register alike. The steam may be gener- 
ated by gas flames or may be taken directly from the engine- 
house boiler. I prefer gas, which is more easily controlled. 

Centrifuges. — Milk and other fluids frequently require cen- 
trifuging, and for this purpose a centrifuge holding at least half 
a liter is necessary. This may be propelled either by steam or 
by electricity. I formerly used a very good electric centrifuge 
made by Lautenschlager but the gearing required so much space 
that I have abandoned it for a much more compact and equally 
serviceable machine made by the International Instrument Com- 
pany, Cambridge, Mass., using electricity as the motive force 
(Fig. 52). Our instrument is so compact that it occupies only 
some waste space behind a door. It is bolted to a thick block 
of concrete (18 by 26 by 30 inches) resting on the floor. It is 



METHODS OF RESEARCH: APPARATUS 



79 



their Type B, Size 2, Amperes 2.15, volts 220. It is 22 inches 
high, the steel shell that encloses the whirling part having a 
depth of 13 inches and an inside diameter of about 23 inches. 
There are 8 carriers (two larger than the others), the total ca- 
pacity being about 1100 cc, and the number of revolutions per 
minute 3000 when accurately leveled, properly loaded and run- 
ning at full speed. The levelling and equal loading are very 
important. 




Fig. 52. — Electric centrifuge made by the International Instrument Company, 

Cambridge, Mass. 

Filters. — The Berkefeld and Chamberland filters are often 
necessary and in connection one must have some kind of device 
for supplying compressed or exhaust air. In vol. I of ''Bac- 
teria in Relation to Plant Diseases," I figured (Plate 10) a 
very good steam pump for furnishing compressed air and a high 
vacuum. Having moved into another building we no longer use 
this particular pump but obtain our exhaust and pressure from 
the main engine room of the Department of Agriculture. Where 
such steam pumps are not available small mercury pumps may 
be used. Very perfect ones are now for sale, of which the Gaede, 
the May-Nelson, and the Geryk are those commonly in use. Of 
the three the Gaede is said to be the best. For removing the 



80 BACTERIAL DISEASES OF PLANTS 

last traces of air quickly from rather large spaces the Langmuir 
Condensation High-vacuum mercury pump is highly recom- 
mended. This requires a rather large opening and the initial 
vacuum must be made by means of another pump. Very 
recently ''A New Cenco High Vacuum Pump" has been adver- 
tised as accomplishing quickly the removal of air from consider- 
able spaces, down to 0.001 mm. without the aid of an accessory 
pump {Science, n.s., Oct. 3, 1919, page x). Its dimensions are: 
length 32 in., width 11 in., height 18 in. Strong claims are also 
made for "The Gramercy Rotary High Vacuum Pump" 
{Science n. s., Dec. 26, 1919 (Cover). 

Titration Apparatus. — See Sutton's "Volumetric Analysis." 
Blood Serum and Starch Media Oven. — See the Text books. 
Miscellaneous. — Grinders, shakers, meat-presses, knives, 
forceps, shears, graduates, beakers, ring-stands, Bunsen burners, 
cork-borers, glass tubing, copper wire and many other small 
articles add to the convenience of the laboratory which should 
be supplied with gas, electricity, hot and cold water, and ice, all 
ir abundance. 

2. For Isolation and Care of Cultures 

Tools. — For isolation of bacteria from plant tissues very 
simple devices are all that is required, viz., steel needles in bone 
or wooden handles, small forceps, spatulas, platinum-iridium 
needles and loops, small knives, scissors, pipettes, glass-tubing, 
rubber-tubing, sterile Petri dishes, test tubes and flasks, a gas 
burner or alcohol lamp, sterile water or bouillon for diluting, and 
suitable culture media. To these may be added large resist- 
ant glass bottles for holding distilled water and stock solutions 
of the standard germicides — especially mercuric chlorid and 
carbolic acid. 

Culture Chambers. — Isolations may be made in clean open 
rooms if air currents are excluded, but it is safer to work under a 
small hood or in a special culture chamber from which the indif- 
ferent and the unclean are carefully excluded. In the Depart- 
ment of Agriculture we use a special standard culture chamber 
made in sections in Baltimore by Ruse and Co., at a cost singly 
of $130 (pre-war price). These are well lighted, of convenient 



METHODS OF RESEARCH! APPARATUS 81 

size and leave little to be desired. They are of polished oak 
(about 10 feet high and with an internal diameter of approxi- 
mately 4 by 4 feet). Specifications and blueprint drawings 
may be had from the Bureau of Plant Industry, United States 
Department of Agriculture. 

The equipment of the transfer room, which should have a 
large window and a broad work-shelf on the side facing the best 
light and several shelves at the right, consists of a cut-ofT gas- 
burner, needles, loops, forceps, litmus paper, and various, mov- 
able racks, tubes and dishes. There should be also a shallow 
drawer under the work shelf for rubber bands, pencils for writ- 
ing on glass, etc. 

Sub-cultures are placed for study at various temperatures. 
These require closed cupboards for room temperatures, ice boxes 
for temperatures from 15°C. to 0°C., electric or gas thermostats 
for temperatures from 30°C. upward, and specially devised 
ammonia apparatus for temperatures below 0°C. 

Thermostats. — Our thermostats are of various patterns. 
Large sizes are preferable. The best one we have is an old in- 
strument covered with felt, made many years ago by Rohrbeck 
in Berlin. All our thermo-regulators are the French metal-bar 
regulator commonly known as the Roux. These require less 
attention than those containing mercury or other fluids. 

Besides ordinary ice boxes we make large use of Paul Alt- 
mann's ten-compartment ice thermostat, and during a part of 
our year can keep the temperature in the lowest compartment 
at 0.5°C., but not during the summer. 

Our stock cultures are carried in ordinary kitchen refrigera- 
tors, but shelves in a specially cooled room would be much better. 
Refrigerators that have the ice compartment over the storage 
chamber are certain to leak into the latter sooner or later and to 
spoil cultures. 

For thermal bath see "Bacteria in Relation to Plant Diseases," 
Vol. I, Fig. 63. 

3. For Preparation and Study of Sections 

A parafhn embedding oven, microtomes, stains, staining 
dishes, flawless slides and covers, good Canada balsam and 



82 



BACTERIAL DISEASES OF PLANTS 



good microscopes are the principal equipment required for the 
preparation and study of permanent sections. 

Microtomes. — Zoologists generally, especially those who 
have studied in Europe, seem to prefer the Jung or the Reichert 
sliding microtome for section cutting. The writer has used both, 
having first learned to cut sections on the Jung, but for many 
years we have used almost exclusively a rotary Dutch machine 
made at Delft and known as the Reinhold-Giltay. Dr. Charles 
E. Bessey was, I believe, the first person in this country to use 




Fig. 53.- — Freezing microtome made by the Spencer Lens Co., Buffalo, X. Y., 
with cylinder of compressed carbon dioxide. 



this machine and his high praise of it induced the writer to order 
one for the Laboratory of Plant Pathology. All that was said 
by Dr. Bessey in praise of this instrument has been more than 
borne out by our experience with it. The machine we first 
bought (for figures of it see "Bacteria in Relation to Plant 
Diseases," Vol. I, 1905, pi. 13 and Fig. 119) is still in use and 
nothing has ever been spent on it for repairs. In recent years, 
with increase of our work, one microtome proved insufficient, 
and we purchased a second machine of the same make. This 
microtome is better, I think, than that similar and more recently 



METHODS OF RESEARCH: APPARATUS 



83 



constructed microtome known as the Minot Rotary, which we 
also have, and use occasionally. For some purposes, such as 
making large sections with a long slant stroke, we have also 
used the large Minot Precision Microtome, which is a very good 
instrument but too cumbrous for our ordinary work. Another 
excellent instrument which we use is the Spencer Rotary Micro- 
tome No. 820. Our freezing microtome also is one made by the 
Spencer Lens Co., of Buffalo, N. Y. 
(Fig. 53). 

The style of razors recommended 
for use with the Reinhold-Giltay 
microtome are figured in "Bacteria 
in Relation to Plant Diseases," Vol. 
I, p. 123. 

Paraffin Oven. — Any small 
water-jacketed oven with a safe, self- 
regulated, constant flame which can 
be set very low will serve for keep- 
ing at the proper temperature (59° 
to 60°C.) the paraffin used in em- 
bedding. For figure of a small 
paraffin oven in use see "Bacteria 
in Relation to Plant Diseases," Vol. 
I, p. 118. This is sufficient for 
private use, but where several per- 
sons must be accommodated at 

the same time the large compartment oven devised by Professor 
Frank R. Lillie is recommended. 

Section Straightener. — For straightening out sections on the 
slides we used formerly the low flame of an alcohol lamp under 
an asbestos board, but more recently we have substituted an 
electric constant temperature apparatus made by W. C. Heraeus, 
G. m. b. H., Hanau a/M., Germany (Fig. 54). With a little 
watching this warm-plate gives a temperature just sufficient 
for straightening the sections without melting them. Previously 
for the same purpose we tried a little electric oven made by The 
Thermo-electric Instrument Co., Newark, N. J. (Freas' Electric 
Incubator, Serial 111, voltage 220, wattage 15.0; 1" X 1" X 




Fig. 54. — Electric warm plato 
used for straightening out para- 
ffin sections. Made by W. C. 
Heraeus. G.m.b.H.. Hanau 
a/M., Germany. Xo. 450. G. 
220 V. 



84 BACTERIAL DISEASES OF PLANTS 

10"; cost $57) but without success, since the temperature proved 
too variable. 

Stains and Staining Jars. — A full set of Griibler's stains 
are very desirable. Few persons know how to use any great 
number of them, but for those few who do they should be avail- 
able. It is best to buy them in unbroken packages. We use 
the Coplin staining jar (figured in ''Bacteria in Relation to Plant 
Diseases," Vol. I, p, 121). There is nothing better than this. 

Mounting Media. — This varies, of course, with the nature of 
the sections. I have not fallen in love with glycerine or glycer- 
ine jelly mounts, but as made by some persons they appear to be 
quite permanent. We employ mostly Canada balsam or Dam- 
mar balsam. It should be purchased only from makers of the 
highest reputation, e.g., Griibler, with special reference to free- 
dom from traces of acids which, if present, will infallibly bleach 
and ruin the best stained slides in course of time. I formerly 
made my own balsam, of good quality, by purchasing the best 
grade of crude balsam and driving off all volatile products in 
an oven kept for some days at the proper temperature, after 
which the brittle unburned residue was dissolved in xylol to 
the proper consistency. I was forced to do this by inability 
to find at that time any fit balsam on the market. 

Microscopes. — ^When properly stained, mounted and dry, 
the sections are ready for study. For this purpose only the best 
microscopes are recommended. At least the objectives, eye- 
pieces, and the substage apparatus should be of the very best 
workmanship, owing to the small size of the objects sought 
and the need of studying them in a clear light with very sharp 
definition. The fine adjustment also should have a very slow 
movement. 

The writer now uses only the Carl Zeiss instruments and 
specially recommends his photomicrographic stand, but the one 
shown in Fig. 55 rather than the newer pattern which is very in- 
convenient to carry, having only two separate awkward finger 
holes in place of the convenient large opening on the old pattern. 
It is also less well finished, e.g., in the two which we have, the 
vernier plate at the right projects above the stage and catches 
the end of the slide, making it very inconvenient to use with 



METHODS OF RESEARCH: APPARATUS 



85 



slides bearing serial sections. These two were made, however, 
since the great German war began, and this may be an incident 
of it. The new pattern differs slightly also in other particulars 
but so far as I can see is not better than the old, if as good. 

Objectives and Eyepieces. — There is nothing better for 
initial magnification than the Zeiss apochromatic objectives, 
especially the superb 16-mm. and 8-mm. dry lenses (each of 
which will give a clear image with a No. 
12 eyepiece) and the 3-mm. 1.30 N. a. 
and 2-mm. 1.30 N. a. homogeneous im- 
mersion objectives, both of which have 
a fairly long working distance, especially 
the 3-mm. 

If one has the 8-mm. apochromatic 
objective and a No. 12 compensating 
ocular, it is seldom necessary to use the 
adjustable-collar Zeiss 4-mm. dry objec- 
tive. We have several of them but they 
cloud easily and, according to my experi- 
ence, seldom give sharp images for any 
great length of time. It is, I think, the 
least useful of all the Zeiss objectives. 
If higher powers than the 16-mm. or 8- 
mm. are necessary, the student should 
learn to use the oil immersion objectives. 
We have also a Zeiss 2-mm. 1.40 N. a. 
and a Zeiss 1.5-mm., but I seldom use 
them. For use with these apochromatic 
objectives several compensating eye- 
pieces are furnished, the most necessary 
of which are the Nos. 4, 8, and 12. For research I generally 
use the No. 8 or 12 eyepiece, the No. 18 is not necessary, and 
for photomicrographic work the No. 4, in place of the special 
eyepieces provided by Zeiss. 

Cheaper microscopes, but very good ones, are also made by 
the Leitz Company, and by the Spencer Lens Company of 
Buffalo, N. Y. 




Fig. 55. — Zeiss Photo- 
micrographic stand and 
small upright camera. 
Nearly all the photo- 
micrographs in this hook 
were made with this 
microscope and camera. 



86 BACTERIAL DISEASES OF PLANTS 

These are the three sorts of microscopes I feel specially 
disposed to recommend. 

Hand Lens. — A good hand lens, that is, one with a flat field 
free from chromatic aberration and having a long working 
distance, is absolutely essential. It should magnify about 6 
times. Hand lenses having a higher magnification ( X 10 or X 15) 
are sometimes very convenient, but are of less general use 
because, owing to their short focus, they will not reach to the 
center of an agar-stab culture or through the top of a Petri 
dish. The writer uses a Zeiss X 6 Aplanat which leaves little to 
be desired. 

4. For Hothouse and Inoculation Experiments 

A large autoclave is necessary very frequently for sterilizing 
pots, soil, discarded infectious plants, and other things used 
about the hothouse. It should be large enough to take in a 
small table, a big inoculation cage, or several 200-lb. sacks 
of soil at the same time. The steam should be from the engine- 
house pipes. The apparatus we use is the Merrel and Soule 
No. 3, made by the Sprague Canning Machinery Company, 
Hoopeston, Illinois, and sold at $45 (pre-war price). Its 
depth is 60 inches and its diameter 45 inches. It is serviceable 
but inconvenient. A larger and more convenient horizontal 
(canners') autoclave, having a depth of 100 inches and an in- 
ternal diameter of 44 inches (Fig. 56) is made by the same firm 
and is called "R K 1. The Rutter Kettle." It is not a very 
perfect autoclave but by attaching to it a small pump to remove 
the air it may be used successfully with steam or may be filled 
with hydrocyanic acid or other insecticidal gases. The Office of 
Seed and Plant Introduction of the United States Department 
of Agriculture has devised several inexpensive convenient pieces 
of apparatus for destroying the nematodes and fungi in green- 
house soil by means of steam heat without puddling the earth. 

Fig. 56. — Sterilization room of Office of Seed and Plant Introduction, United 
States Department of Agriculture, Washington, D. C. The Rutter Steam Kettle 
in the center. At the extreme left is the pump for removing air from the auto- 
clave or gas, if used as an insecticidal chamber. (Courtesy of Dr. B. T. Galloway.) 



METHODS OF RESEARCH: APPARATUS 



87 




88 



BACTERIAL DISEASES OF PLANTS 




Yu; 57 —Home-made device for steaming infected soil. It is a steam-chamber 
holding 12 drawers having wire-mesh bottoms. These slide in and out on rollers. 



METHODS OF RESEARCH: APPARATUS 89 

One simple piece, a sort of tall deep cupboard, which any car- 
penter can make and which takes up very little room, will 
sterilize 6 bushels of soil in an hour (Fig. 57). This earth is 
held in half-bushel lots resting on sacking in shallow drawers 
having wire-mesh bottoms. The steam enters at the bottom 
(back) and the air flows out at the top (front) through openings 
which are closed by a slide when steam begins to escape. The 
cupboard is 3 inches deeper than the length of the drawers and 
every other one of these is shoved back so that the steam (15 
pounds pressure) readily passes under and over each drawer. 
The later are 18 inches wide, 30 inches long and 3 inches deep. 

The other pieces of hothouse apparatus required are very 
simple. 

In addition to knives, labels, steel needles, hypodermic 
syringes (rarely necessary) and atomizing devices (a cologne 
sprayer will do), I need mention only alcohol lamps, cages with 
walls of glass (inoculation cages) and cages with walls of wire- 
gauze (insect cages). These may be made of such size as will 
suit the best convenience of the experimenter. One whole 
side should be used as an opening (double door), and for the 
insect cages an additional small opening is very desirable. The 
bottom of each is, of course, open, resting on the earth, or on a 
bench or platform of some sort. 

5. For the Photographic Room 

If possible, the photographic room should have north, 
south, and overhead hght, or at least south light and overhead 
north light in order that advantage may be taken of direct 
sunlight for heliostats, etc., and overhead hght for a variety 
of indoor photographic lightings. For these reasons it is best 
located on a top floor. Our own rooms being on a middle floor 
we have had to make shift with south light only, taking it from 



and are shorter than the depth of the chamber by a few inches allowing every other 
one to be shoved back or pulled forward so that steam, which enters at the back, 
at the bottom, may circulate freely around each one. The soil is placed on coarse 
sacking and each drawer holds a half bushel. Air is displaced through the two 
holes at the top and these are closed by slides when steam begins to appear. Photo- 
graph from Dr. B. T. Galloway. 



90 BACTERIAL DISEASES OF PLANTS 

two big windows. One window is hardly sufficient for such a 
room if several persons are to use it, since it is often necessary 
to make natural size pictures and photomicrographs or lantern 
slides at the same time. For photomicrographs I use southwest 
light in the morning and southeast light in the afternoon, pro- 
tecting the camera from the direct light of the sun, the south 
window therefore must not be too close to either wall. 

The principal pieces of apparatus in our room are the camera 
stands, two or three small tables and wall-cases, and the sink 
and washing boxes. It can be entered without passing through 
the anti-chamber of the dark room. This is important. 

Cameras and Lenses. — Whatever results are worth the time 
spent on them are also worth recording, therefore, from the be- 
ginning the student who hopes to become a naturalist should be 
taught to use the camera as freely as the microscope. If he 
learns to do this early he will have a great advantage over such 
as neglect it. The essentials are a light-tight bellows and a 
lens that will give a correct and sharp image, to w^hich has been 
fitted a shutter allowing for measured long and short exposures. 
Shutters, however, are not absolutely essential and the size of 
the camera is also of minor importance, if the image is sharp 
enough to enlarge, as it will be, provided the lens is good, and 
the focus for the photograph is correct. If, however, the lens is 
poor or the focus is nearly out, the picture will not stand en- 
largement. The writer with Zeiss lenses has made pictures 
sharp enough to stand a X 10 enlargement. The camera for 
natural size work should have a bellows long enough to give a 
magnification of two diameters, i.e., it should be 3 times the 
equivalent focus of the lens. I have seen nothing better than the 
Folmer and Schwing (Eastman Kodak Company) Q}^ by 83-^ 
slightly modified Collins and Brown camera figured in Vol. I 
of "Bacteria in Relation to Plant Diseases" (pp. 134 and 145). 
For a light, traveling camera provided with a tripod, the Eastman 
Company Century Grand (4 by 5) leaves little to be desired. 
This is much less bulky than the Folmer and Schwing 5 by 7 
Reversible-back Graphic, which, however, is extremely compact 
and durable, and in weight stands about midway between the 
two already mentioned. The lenses for such cameras should be 
the best on the market, and should be provided with the Volute 



METHODS OF RESEARCH: APPARATUS 



91 



shutter. I prefer the Zeiss double-protar lenses, Series Vila. 
These are made and sold by Bausch and Lomb, Rochester, N. Y., 
under the name of Convertible Anastigmatic Lenses, Series 
Vila. Very recently (1919) Mr. E. L. Crandall of the United 




B 



i^ 




Fig. 58. — The Crandall model view camera which can also be used for small 
objects natural size or X 2. Bellows extension 18 inches, weight 53-^ pounds with- 
out tripod. Well made. This takes a B. and L. Zeiss Double Protar Vila lens 
having a focus of about 5 inches. Front combination focus about 8 inches, rear 
combination focus about 12 inches. Made by the Folmer and Schwing Division 
of the Eastman Kodak Co., Rochester, N. Y. It carries a film, dry plate or Lu- 
meire plate 3'^ by -i^i inches. 



States Department of Agriculture, has designed an excellent, 
moderate priced small camera for field work that can also be 
used for small objects natural size or twice enlarged (Fig. 58). 
This also is made by the Eastman Kodak Co. 



92 BACTERIAL DISEASES OF PLANTS 

The Zeiss Planar lenses, Series la, may have a word here. 
They are fully described in the Zeiss catalogues and I have 
figured Nos. 1 to 5 (those best adapted to photomicrographic 
work) in "Bacteria in Relation to Plant Diseases," Vol. I, p. 132, 
together wdth the special substage condensers to be used with 
them. These lenses have a focus varying from 20 mm. to 100 
mm. and yield, when stopped-down, images ranging from 1 to 
5 inches in diameter. They may be used either on the micro- 
scope or on the ordinary camera. They are very rapid lenses 
working with a minimum of light. They yield very sharp 
images, but have not much depth of focus. As stated in the 
catalogue "Focusing must be done with the most scrupulous 
care." I generally use a hand lens, focusing on a clear-glass 
image, and stop-down as much as possible to get all the depth 
of focus possible. In selecting a focus with the stop wide open 
it is well to remember that the increased depth of sharpness 
to be obtained later by stopping-down will be mostly away 
from the observer rather than toward him. 

Camera Stands. — For many purposes a simple tripod, or a 
home-made device such as that figured in "Bacteria in Relation 
to Plant Diseases," Vol. I, p. 133, is sufficient, but for use with 
planars and generally for best results in lighting, we now use 
a universal position stand, i.e., the stand made by Folmer 
and Schwing (Eastman's catalogue of photographic apparatus 
and materials, p. 50) with important modifications by James 
F. Brewer, as shown in Figs. 59-60. These improvements 
consist of (1) a device at the bottom of the stand for rotation; 
(2) castors for translocation ; (3) a swing device under the back- 
board for raising or lowering the camera as a whole and locking 
it in the position desired; (4) an object carrier movable not only 
backward and forward but also to either side and away from 
the central axis of the stand; (5) a carrier behind the preceding 
for the background; (6) a screw device for shortening or lengthen- 
ing the distance of the camera as a whole from the object; 
(7) a test-tube rack attached to the front part of the object 
carrier and sliding up and down. In this carrier are a series 
of inch holes through which are thrust the tubes to be photo- 
graphed, the same being held in place by a lining of perforated 



METHODS OF RESEARCH: APPARATUS 



93 




Fig. 59. — Camera stand used by the Laboratory of Plant Pathology. Made by 
Folmer and Schwing Division, Eastman Kodak Co., Rochester, N. Y. 




Fic. 60. — Horizontal arrangement of Fig. 59. 



94 BACTERIAL DISEASES OF PLANTS 

rubber, the holes in which are smaller than any test tube but 
are slit in four places 90° apart so as to receive and hold tubes 
of any size. For very large tubes a second carrier is provided. 
With these additions the apparatus is most convenient since 
it can be rotated toward any part of the compass, tilted and 
locked at any angle from vertical to horizontal, lengthened 
or shortened as required, and the various parts shifted so that 
the object to be photographed can be centered on the ground 
glass with a minimum of labor. Two additional changes 
would make it nearly perfect — (1) a cut in the top of the frame- 
work at a given point (x) so that the object carrier might be 
removed (lifted out bodily) without sliding it all the way to the 
end of the frame-work, where the background carrier is often 
in place and must now also be removed to let it out; (2) the 
object carrier can be moved to the right or left any distance 
desired, but only 3 inches away from the central axis of the 
stand, whereas sometimes it is desirable to move it farther, that 
is, 4, 5, or 6 inches. This could be accomplished very easily 
by lengthening the stop groove in the brass strap on the under 
surface of the frame. 

Photomicrographic Work. — Nearly all of the photomicro- 
graphs used in this book were made on the small Zeiss upright 
camera (Fig. 55). (For more details see ''Bacteria in Relation 
to Plant Diseases," Vol. I, and various text-books.) Dr. L. B. 
Wilson of the Mayo Laboratories, Rochester, Minn., has de- 
vised and described a very convenient upright stand. For an 
illustration of this photomicrographic camera see the 1917 cata- 
logue of the Spencer Lens Co., Buffalo, N. Y., p. 120. 

Light Filters. — For the large horizontal photomicrographic 
camera I have generally used the green, fluid, ray-filter known 
as the Zettnow filter, but for planars the dry Wratten filters 
made by the Eastman Kodak Company, Rochester, N. Y., 
are very useful. (Catalogue and explanations for use will be 
furnished by the Eastman Company on application.) 

Dry Plates and Developers. — All these have been greatly 
improved in recent years. The essentials in dry plates and films 
are to have some giving strong contrasts and others correct 
color values. It is sometimes impossible to tell in advance 



METHODS OF RESEARCH: APPARATUS 95 

whether an ordinary plate or an orthochromatic plate will give 
best results, colors having much the same appeal to the eye 
sometimes behaving differently on the dry plate. The writer 
uses Seed's rapid, gilt-edge (non-isochromatic) plates for some 
purposes and for others Cramer's instantaneous and slow iso- 
chromatic plates, also Hammer's non-halation (double coated) 
orthochromatic plates, and for exact color values Wratten and 
Wainwright's Process Panchromatic plates, these last being very 
sensitive even to dull red light. 

Any one of half a dozen developers may be used. It is good 
to stick to one until you have mastered it. We use at present 
Citol which has the advantage of great simplicity, the only 
preliminary to its use being its dilution with 20 parts of distilled 
water, more or less, according as the plate is under or over 
exposed. For contrasts in photomicrography I use a hyclro- 
chinon developer. 

Washing Devices. — Two washing tanks made by Burke and 
James, (Jackson Blvd. and Desplaines St., Chicago, 111.), have 
proved very useful. The one for negatives takes any size 
without readjustment, and jets currents of water over the nega- 
tives from two directions, thus insuring a very thorough circu- 
lation and removal of the fixative in a minimum time. The one 
for washing prints also has an ingenious device for keeping the 
water in circulation and the prints separated and moving about. 
They should be made of tinned copper, but unless specially so 
ordered they are made of galvanized sheet iron which soon rusts 
out. 

The Developing or Dark-room. — The dark-room should be 
impervious to light. If it is set up in part of a larger room 
after the construction of the building, the walls may be of inch 
pine boards, matched, and should be covered inside with heavy 
carton (composition) board which must be painted a dead (lus- 
terless) black. 

By preference, the entrance should be through a labyrinth 
(a reversed and flattened letter S, or if one thinks of the walls 
rather than of the passage way, then two linked letter U's (E)) 
opening outward into the darker part of an ante-room. Doors 
are inconvenient, and if they open directly into the dark-room 



96 



BACTERIAL DISEASES OF PLANTS 



from a light room they must be kept locked while development is 
going on. Often this is very inconvenient to others, but it 
is the only safe way. 

Our own dark-room (Fig. 61) occupies the northwest corner 
of a well-lighted large south room which is used for general 
photographic purposes. It measures, inside, approximately 9 
feet north and south bv 15 feet east and west, and is divided into 




-2'- -J. 

050: 



PR/NT/NG 
/SOOM 




DARK 



HOOM 



- -3 10"- - - 
LOADING 



3' 2' +-14- 

TABLE 



+ t 

36^ - - 

SINK 



2 ,,-H 



TABLE 
O 



O O 
O O 



O ELECTRIC 8ULB5 

-r TAUCETS 

. .RUNWAYS 

^WINDOWS IDARK RO0M,19 j2 6 ) 

i ELECTRIC SWITCHES 



PMOrOGMPN/C £00A/ 



D 



WINDOW 

- S -^ 



u 



CAMtRAS 

WINDOW 




Fig. 61. — Diagram of photographic room, printing room and dark-room used by 

the writer. 



two parts: (1) an ante-room on the west side, used for making 
prints; (2) the dark-room proper, used only for the development 
of negatives. The ante-room is long and narrow with a sink 
at the northwest corner (behind the corridor door), the re- 
mainder of the west wall space being occupied by a printing and 
developing table. The printing is done by means of a battery 
of large size, fixed tungsten lights, in the southwest corner. The 



METHODS OF RESEARCH: APPARATUS 97 

developing is done under a red (bulb) drop light near the sink. 
There is a door at either end (both opening inward) : one gives 
entrance from the bright photographic room, the other from the 
rather dark central corridor of the building. Both should be 
hinged on the side nearest to the developing table. The size 
of this ante-room is 5 feet 8 inches by 9 feet. 

The dark-room proper takes the remainder of the space (9 
feet 4 inches by 9 feet), but one corner of it is occupied by the 
labyrinth so that the actual working space is considerably 
reduced. The lab^Tinth (2 feet ^^ide) begins in the northeast 
corner of the ante-room next the door opening into the dark 
corridor. As an additional precaution against the accidental 
entrance of light, this corridor door is provided with a push 
lock to be used whenever necessary. On the right-hand side, 
as one enters the dark-room proper, behind the south wall of the 
labjTinth, is a narrow space occupied its whole length on one side 
(the north side) by the loading shelf. On the east wall, facing 
the observer as he enters the dark-room, is the developing shelf. 
At his left against the north (corridor) wall is the fixing shelf 
and in the angle between the two shelves is the deep sink (36 
inches long by 20 inches wide). The dark-room consists, 
therefore, of two rectangles, the larger o by 9 feet occupied on 
the outer (east) side by the long developmg shelf and the sink 
just referred to; and at one end (the north) by the shorter fixing 
shelf, and at the other (south) end opening into the smaller 
rectangle (3 feet 10 inches long by 3 feet wide) which is occupied, 
as already stated, bj^ the loading shelf. This abuts against the 
labyrinth wall, and above and below it are other shelves for the 
storage of boxes of dry plates and plate holders. There are also 
shelves under the developing shelf for extra trays, and under and 
above the fixing shelf. 

Two persons can develop in tliis dark-room without inter- 
ference and the loading shelf is also long enough to allow two 
persons to use it at the same time. There is. however, no waste 
space and everytliing is witliin convenient reach. A step or two 
brings you to any part of the room. 

The subdued fight by means of which exposures must be 
developed may be sunhght or electric fight, passed through a 



98 BACTERIAL DISEASES OF PLANTS 

series of screens, i.e., ground glass, orange glass or orange paper 
and red glass. An opaque screen also should be provided and 
all the screens should be counter-weighted to slide up and down 
easily inside a window frame. There should be at least two 
of these windows. Our room has three. Two are over the 
developing shelf and a third is in the south wall between the 
developing shelf and the loading shelf. 

The developing shelf should face the source of light at a 
convenient height rather than receive its light from one side. 

The washing sink (1 foot deep) should be close to the de- 
veloping shelf to avoid waste of time and on the other side of 
it should be a roomy fixing shelf. By ''roomy" I mean large 
enough for several trays. 

The loading and fixing shelves and the developing table are 
each 36 inches high. The loading and fixing shelves are 18 
and 16 inches wide. The developing table is 20 inches wide and 
the surface of this table consists of two removable frames of 
slat-work. Under it is a shallow lead-lined sink sloping to the 
left and emptying into the deep sink. The window-ledges aye 
Sj-i inches above the top of this table. 

There should be plenty of storage shelves in the dark-room 
(under the fixing shelf and the developing shelf, and over them 
also) for trays, bottles, beakers, graduates, etc. 

The loading shelf should be in the darkest part of the room, 
i.e., as far away from the red lights as possible and behind the 
door or labyrinth, so that there shall be a minimum of danger 
from fogging when boxes of dry plates are opened. I open boxes 
of dry plates, always, with the opaque screen drawn low over 
the red light, especially if they are orthochromatic plates. 
The washing and printing are best done in adjoining rooms 
(see Fig. 61). Dark-rooms, like kitchens, are most convenient 
if everything is handy; they should not, therefore, be very large, 
and consequently some sort of ventilation becomes necessary, 
especially if they are much used. In the top of our own room 
I have inserted a hood in the bottom of which is placed an electric 
fan which can be turned on at will and which rapidly pumps the 
foul air out of the room, the fresh outside air flowing in through 



METHODS OF RESEARCH: APPARATUS 99 

the labyrinth. This cost only a small sum and has proved 
very satisfactory. 

The electric bulbs for the red lights are outside but may be 
turned on or off without leaving the dark-room, by means of a 
double push button at Z (Fig. 61). 



USES OF CULTURE-MEDIA 

Culture-media are needed every day in the routine work 
of the laboratory and are required for several distinct purposes : 
(1) for the isolation of organisms from mixtures or directly from 
diseased tissues ; (2) for the long-continued growth of organisms 
without loss of virulence; (3) for differential descriptive pur- 
poses; (4) for cultures adapted to chemical analysis. 

For the first purpose we must study the nature and needs 
of the various parasites, and when they differ from the common 
sorts must cater to them, devising media exactly suited to their 
requirements, or at least better adapted to the needs of the para- 
site than to those of the accompanying saprophytes. This 
frequently requires considerable study, as to range of toleration 
of acids, alkalies, salts, N.-compounds, sugars, alcohols, etc., 
but standard media should be tried first. For first isolations I 
always try +15 peptone-beef-agar poured plates. If this fails, 
then +7 beef -peptone agar may be tried, and other media, such 
as steamed potato and special agars, e.g., dextrose potato agar 
or whey agar. 

The second kind of media varies a great deal with the or- 
ganism and can be discovered only after prolonged study of the 
parasite on a variety of substrata. Some observations on such 
media will be found under the various diseases described in 
Part III. 

The third sort need not be, and in fact cannot be, media of 
universal value. They are good only for the particular purpose 
in mind, and the future will see a large increase in their number. 
What we seek here are media that will bring out not necessarily 
good growth, or any growth at all, for that matter, but dijfer- 
ences in behavior when a variety of bacteria are tested in it, that 
is, changes in gross appearance, morphology, pigmentation, pre- 
cipitates, pellicles, crystals, weak vs. dense clouding, medium 



100 BACTERIAL DISEASES OF PLANTS 

reactions (acid, neutral, alkaline), using neutral litmus and phe- 
nolphthalein, etc. — and here a medium generally neglected by 
bacteriologists may be just the one needed. The student should 
not regard the chart sanctioned by the Society of American 
Bacteriologists as in any sense a finality — there are no last words 
in science, at least not in bacteriology — and he must be always 
on the lookout for simple and effective means of differentiation. 
Often, when the colonies of two organisms look exactly alike on 
+ 15 peptone-beef agar, we try potato agar, prune agar, string- 
bean agar, starch agar, whey agar, or some kind of gelatin 
medium, and find a difference. In this connection see Fig. 184. 

For the fourth purpose, to avoid complications, one would 
naturally select, first of all, well-aerated, simple synthetic media. 

Carefully considered (tested) formulae for the preparation 
of various culture-media will be found in '' Bacteria in Relation 
to Plant Diseases," Vol. I. For Meyer's mineral solution con- 
sult Ibid., Vol. Ill, p. 250. 

PREPARATION OF CULTURE-MEDIA 

It is impossible to pursue the study of bacterial diseases 
of plants without the use of at least some forms of culture media, 
and the student should know how to prepare with his own hands 
all necessary substrata. It is a useful training of the hands 
and of the judgment even for those who are not to use it later 
in research. 

Culture-media may be divided into two classes: (1) complex 
organic substances and (2) simple synthetic preparations. The 
first, though they are harder to prepare, are still used more 
generally than the second, and are in the main better adapted 
to the growth of parasitic micro-organisms than are the more 
exact media compounded out of simple chemical substances. 
The latter, however, may be expected eventually to take the place 
largely of the more or less variable animal and plant compounds 
now in general use. At the same time the writer would like 
to register an objection against the discontinuing of any of the 
cultural substances now in use until such time as we have well- 
recognized suitable substitutes. At present we need all of them, 



METHODS OF RESEARCH: PREPARATION OF CULTURE MEDIA 101 

especially for the study of parasites. Before proceeding to a 
discussion of these cultural substances, the student should read 
quite carefullythat part of the preceding chapter on Apparatus, 
and also what he can find on the making of culture media in 
other text books. 

In the preparation of culture-media one must be governed by 
the substances and the apparatus at hand. The simplest sub- 
stances to prepare are cylinders and slices of potato, carrot, 
and other vegetables, and these are still of great use in the 
study of diseases of plants, and not to be discarded for purely 
synthetic media. These vegetable substances may be used 
either cooked or raw. If cooked, they are best kept in cotton- 
plugged test tubes in the form of slant cylinders, standing with 
the bottom immersed in a small quantity of water or free on 
wet cotton. If raw, they may be cut into the form of cubes or 
slabs and placed in deep Petri dishes. We will first discuss the 
proper preparation of such media. A great variety of cooked 
vegetable substances can be used, both for study of pure sub- 
cultures of the various micro-organisms, and in default of gelatin 
or agar for their isolation from the diseased plants. We will, 
therefore, first consider the proper preparation of these sub- 
stances. 

Steam-sterilized Solid Vegetable Substances. — Select sound 
carrot roots, potato tubers, turnips roots, and similar vegetables, 
wash thoroughly under the tap with hand-rubbing or brush- 
scrubbing to free from dirt, and with a clean knife pare away 
the outer surface, being careful to contaminate the inner por- 
tions as little as possible by contact with the outer surface 
of the parings or with soiled hands. The remainder may 
now be thrown into beakers of sterile water and rinsed, after 
which cylinders may be punched out with a nickel-plated cork 
borer of the proper size, slanted with a sharp knife and, after 
a preliminary rinsing, dropped by means of sterile forceps into 
clean test tubes after which the necessary amount of water 
should be added and the tubes plugged with clean cotton. The 
cylinders should be smaller than the bore of the test tube so 
that there may be room for the water, otherwise they soon dry 
out and are not then suited for the growth of many organisms. 



102 BACTERIAL DISEASES OF '^PLANTS 

There should be enough water added so that the culture will 
remain moist for a number of weeks but not enough so that the 
slant surface is covered. For isolation purposes, by means of 
parallel strokes, the slant should be long and the amount of 
water such that it will in no case flood over the slant surface. 
Merely for the study of the organism on a particular medium 
this is not so necessary. When the tubes have been filled and 
plugged they must be steamed on 3 consecutive days, for 15 
minutes each time. In default of a steamer they may be 
stood upright in a tea-kettle and boiled. Formerly we had much 
trouble in this laboratory in sterilizing potatoes and similar 
substances derived from roots, i.e., after three steamings the 
cultures would often develop a wrinkled white or gray white 
bacterial growth. This growth came from spore-bearing bac- 
teria lodged on the surface of the potato tuber and introduced 
into our .tubes through defective technic in their preparation, i.e., 
we were not careful enough in paring and handling the potatoes 
and other vegetables used, the surface of which almost always 
contains resistant spore-bearing bacteria. We are now very 
particular in the preparation of these media not to contaminate 
either the pared portions or the cylinders punched from the 
same. For that reason we rarely handle them with our fingers 
in the last stages, but with forceps, and before putting them into 
the tubes we give them a final rinsing in beakers of distilled 
water, and now generally we autoclave such media. To avoid 
erroneous conclusions it is well to let the sterilized medium 
remain on the shelf some days before using it, that the unkilled 
spores of fungi and bacteria may germinate, and always, of 
course, each tube should be critically inspected before it is in- 
oculated. In some laboratories it is difficult to keep media 
sterile because Penicilliums and other common fungi have been 
allowed to fruit freely in the rooms until the whole laboratory 
has become an unclean place. Such laboratories are a disgrace 
to the profession. The first thing is to have a general cleaning 
up, and then to stay clean. In 1917 we had much trouble for 
some weeks from a spore-bearing, very heat-resistant and acid- 
resistant white schizomycete introduced from the Middle West 
on dirty culture dishes. 



METHODS OF EESEARCH : PREPARATION OF CULTURE MEDIA 103 

Preparation of Raw Vegetable Substances. — Select, as 
before, sound tubers and roots, etc., wash thoroughly with 
hand-rubbing or brush-scrubbing. Instead of paring, plunge 
them one minute into alcohol (to drive air out of the crevices), 
and then 30 to 40 minutes in 1 : 1000 mercuric chlorid water, 
then remove, drj^ with a sterile cloth, or with sterile filter paper, 
and cut on a sterile surface into suitable slabs with a cold sterile 
knife; one of large size such as is used in kitchens is suitable. 
As soon as cut, the slices are removed without touching the 
cut surface and placed in sterile deep culture dishes in pairs, one 
of w^hich may be inoculated and the other held as a check. 
If this w^ork is done in clean still air, with sterile instruments, 
and if the cut surface is never touched with the hands or with 
contaminating instruments, there will be little opportunity for 
intruding organisms to obtain a foothold. Undoubtedly some 
bacteria on the roots are not killed even by the long soaking 
in mercuric chloride water but their surface layers are so im- 
pregnated with the poison that they seldom develop or give 
any trouble in the dishes. The amount of poison dragged across 
the cut surface in making the slices is wholly negligible, if the 
roots are dry when cut. 

Fluid Vegetable Substances. — The juices of vegetables 
diluted with water are also useful as culture-media. They may 
be used in 10-cc. portions in test tubes, or filled into suitable 
fermentation tubes, or used in flasks. These are generally 
steam-heated a short time on 3 consecutive days before using. 
The same care should be used in the preliminary surface prepa- 
ration of vegetables, the juice of which is to be used for culture- 
media as has been recommended already for the preparation of 
steam-sterilized potato cylinders, etc. This juice is obtained 
either by cooking or by crushing or grinding and pressure. It is 
very easy to sterilize such media if they have not been con- 
taminated by carelessness in the initial stages of the prepara- 
tion. These fluids must be filtered before they are tubed, 
first through two folds of cheesecloth or surgeon's gauze and 
afterward through filter paper. Substances containing starch 
filter with difficulty after they have been boiled. Therefore, 
if juices are to be extracted from starchy vegetables by heat 



104 BACTERIAL DISEASES OF PLANTS 

in advance of filtration it is desirable that such heating should 
not be carried to the boiling point. We extract potato juice 
from thin slices of the tubers by heating for an hour in double 
their weight of water at a temperature not higher than 60° C, 
in order that we may be able to filter the fluid without difficulty. 
Fluids extracted from vegetables by grinding and pressure are 
usually not very well adapted in their concentrated form to 
the growth of bacteria and must, therefore, be diluted with a 
considerable volume of water before they are steam sterilized 
(1:4, 1:10 may be taken as standard dilutions, but I also try 
them full strength). Such juices may be sterilized cold, if de- 
sired, by passing them through a Chamberland filter free from 
cracks. 

Animal Substances. — Cow's milk is one of the easiest and 
most satisfactory of animal substances for use, provided it can 
be obtained in a fresh condition and is properly sterilized. I 
use it both with and without cream, especially without. For 
separating the cream we use a centrifuge. Small quantities 
may, however, be prepared nearly cream-free by filtration 
through coarse filter paper but this is a slow process. Over- 
heating changes the nature of milk somewhat and should be 
avoided. We generally use it in 10-cc. portions in sterile test 
tubes, cotton-plugged, which portions are steamed on 4 consecu- 
tive days, each time for a short period only. Often three times 
heating is enough, but occasionally the fourth heating is re- 
quired to render the milk free from resistant bacteria, and there- 
fore we have adopted the four heatings, as a routine practice. 
These steamings may be for 15 minutes on the first day, 10 min- 
utes on the second and third days, and 5 minutes on the fourth 
day. This destroys all aerobes, but some anaerobes may re- 
main. Milk should not only be used in the manner described 
but should also be used in the form of litmus milk, only enough 
sensitive blue litmus being added to render the fluid a lilac or 
lavender blue. If the curd is precipitated in the steaming, then 
something is wrong either with the litmus or with the milk. 
Usually it means that such milk is old and full of organisms and 
their by-products. Methylene blue may also be added to milk 
(6 cc. of 1 per cent, methylene blue in distilled water to each 
200 cc. of milk) for study of the reducing activities of organ- 



METHODS OF RESEAECH: PREPARATION OF CULTURE MEDIA 105 

isms. It is best not to autoclave milk or any substance contain- 
ing sugars. We do, however, autoclave most other substances, 
heating them seldom higher than 110°C. or longer than 15 or 
20 minutes. 

Next to milk, beef bouillon is one of the most commonly- 
used substances in the bacteriological laboratory. It may be 
used alone but is generally fortified by the addition of 1 per cent 
peptone^ to which sugar is also sometimes added. A number 
of peptones are on the market and they vary so much in quality 
and in their nutrient value for organisms, that it is best, except 
for special experiments, to make use of only one, namely, that now 
commonly recommended in bacteriological laboratories, and 
known as Witte's peptonum siccum (See the observation under 
Olive Tubercle Organism, No. XIII, concerning the inhibiting 
action of Merck's peptone from flesh). Beef bouillon is much 
better, in my judgment, if made from flesh of beef than from meat 
extracts. In my experience meat extracts often contain salt and 
bacteria which are hard to sterilize in the steamer, whereas a 
good grade of fresh meat seldom gives any trouble. For further 
notes on preparation of this medium and many other media 
consult "Bacteria in Relation to Plant Diseases," Vol. I. 

Blood Serum. — Loffler's solidified blood serum is another sub- 
stance much used by the bacteriologist, but as it is rather dif- 
ficult of preparation, for those who do not have access to the 
apparatus and the animals of an animal pathological laboratory, 
it is recommended to purchase from time to time in the open 
market the small amount the plant pathologist needs. We buy 
ours from Parke, Davis and Company, Detroit, Michigan. 

Gelatin and Agar Media. — The bacteriologist makes very 
extensive use of gelatin and agar culture media in the form of 
Petri-dish poured plates for isolation of his organisms. The 
basis is peptonized beef bouillon to which agar is generally added 
in 1 per cent or gelatin in 10 per cent quantities to obtain the 
proper solidity. Of course, other substances may be employed, 
if desired, instead of beef juice, such as simple peptone-water 
or peptone-water reinforced with 1 or 2 per cent cane-sugar, 
grape-sugar, milk-sugar, etc. On the whole, agar is more useful 
for isolation purposes than gelatin, especially in very warm 



106 BACTEEIAL DISEASES OF PLANTS 

climates. The student, therefore, must learn how to prepare it. 
For details of preparation of both agar and gelatin, consult 
''Bacteria in Relation to Plant Diseases," Vol. I, pp. 29 to 36. 
Our Standard agar is +15 and our Standard gelatin +10 on 
Fuller's scale, or 1.5 per cent and 1 per cent respectively, 
if reckoned on 100-cc. portions. It is better to keep to Fuller's 
scale since we make up media in liters not in 100 cc. portions. 

We still use Nelson's photographic shredded gelatin for our 
gelatin culture media and for our agar media a powdered agar 
made by Kahlbaum in Berlin, and have no trouble in filtering 
either one or in obtaining a uniform and clear product. Diffi- 
culties of agar filtration may usually be traced to the fact that 
the medium has not been cooked sufficiently. The work must 
then be done over. It is a lazy bacteriologist who is content 
with a clouded culture medium, either fluid or solid. 

Peptone -water Media. — For the study of the behavior of 
organisms in contact with various sugars and alcohols we use 
distilled or river water to which has been added 1 or 2 per cent 
Witte's peptone and the desired amount of the carbon food, 
seldom more than 2 per cent. It is steamed, filtered, filled into 
fermentation tubes and sterilized discontinuously as in the case 
of other substances. Comparison should be made using other 
peptones, Merck's, Difco, etc. 

Excess of acidity or alkalinity of medium should be corrected 
by determining the actual acidity or alkalinity by titration, 
using phenolphthalein and N/20 sodium hydrate or N/20 hydro- 
chloric acid, as the case may be, and then adding calculated 
quantities of a much stronger acid or alkali and re-titrating. 
The latter must not be neglected. Without titration it is impos- 
sible to have any two batches of media alike, but when it is regu- 
larly employed there is great uniformity in the product of the 
laboratory, and in the results obtained. Our method is to put 
50 cc. of boiling distilled water in a white capsule with 3-^ cc. of 
the phenolphthalein solution and 5 cc. of the substance to be 
tested and run in barely enough alkali to give a trace of pink color 
which is the neutral point. Do not re-boil, nor make the solution 
red. The slightest pink that can be seen is the place to stop. 

Synthetic Media. — Certain synthetic media have great use 
in the bacteriological laboratory because micro-organisms be- 



METHODS OF RESEARCH: PREPARATION OF CULTURE MEDIA 107 

have toward such media in quite different manners. Cohn's 
solution, Uschinsky's solution, Fermi's solution and Meyer's 
solution are examples of such media ; for the preparation of these 
and others consult ''Bacteria in Relation to Plant Diseases," 
Vol. I and the various text books. 

Pure Chemicals. — The best quality of everything, without 
regard to expense, should be the rule. Especially in the prepa- 
ration of synthetic media and in fermentation studies must 
absolute purity of the chemicals be assured. Pure sugars and 
alcohols, in particular, are difficult to obtain, and expensive. 
Rare sugars are now to be had in this country from Digestive 
Ferments Co., Detroit, Mich., and the Special Chemicals Co., 
Highland Park, Chicago, 111. 

TECHNIC OF ISOLATION 

I have said so much on this subject under the different dis- 
eases in Part III, that not much need be said here, particularly 
if the student reads carefully what is said on this subject in the 
various text books recommended for his perusal on page 76. 

Fire. — This is a very certain method for disposing of trouble- 
some bacteria and is often resorted to by the bacteriologist. 
"Flamhez vos vases, flamhez vos vases!'' cried Pasteur on a memo- 
rable occasion. With the same end in view, flame, or burn 
lightly with a hot spatula, the surface from beneath which you 
wish to take material for a transfer. A little experience will 
be more useful to you than many words. You should aim 
to scorch the surface without cooking the interior. It is best, 
however, to err on the side of underheating. 

Autoclave all discarded culture dishes before the glassware 
is handed over to the servant to be washed. 

Germicides. — Certain chemicals, in another way, bring 
about the same result as fire. Often, however, I use these 
substances so that they act only as antiseptics. How long they 
may be allowed to act and how strong they may be used, de- 
pends on the nature of the germicide, and on the character of the 
tissues exposed to it. We use about the laboratory commonly 
only two germicides, 1 : 1000 mercuric chlorid water, and 5 



108 BACTERIAL DISEASES OF PLANTS 

per c^nt carbolic acid water, the former more than the latter. 
Surfaces covered with a thick cork layer, e.g., potato tubers, 
stand exposure to 1 : 1000 mercuric chlorid well (30 minutes); 
thin green parts, such as pieces of leaves, should be exposed 
to it only for a minute or two. Infected tools which cannot be 
flamed may be dipped into the carbolic acid water. Carbolic 
acid should not be used on the hands. Discarded culture-tubes, 
flasks, dishes, etc., which are not autoclaved should be treated 
with the chromic acid cleaning mixture before washing. Rubber 
gloves should be used in handling this mixture. Infected hands 
may be washed in the mercuric chlorid water. The student 
should remember that germicides are poisons, and should 
govern himself accordingly. They must not be left where 
children or animals can get them. Hands accidentally wetted 
with the chromic acid mixture should be washed immediately 
and thoroughly. 

Still Air. — I regard clean still air as very important. For 
this reason I never make transfers in the open room, but always 
under a hood or in a special transfer chamber, such as I have 
described, and I do not advise its ventilation by means of an 
air current. 

Clean Hands. — These are still more important. The aver- 
age individual infects everything he touches, and also, as a rule, 
touches everything within his reach. Hence the great prevalence 
of contagious diseases! Apparently it is a necessary part of 
the psychology of a great many persons to become satisfac- 
torily acquainted with an object only through the sense of touch. 
Deprived of touch such persons are almost as blind as the sight- 
less. Until the student has learned to consider his hands, even 
when "clean," as never really clean but always as contaminated, 
he has scarcely made a beginning in bacteriology. Your hands 
should be kept out of media and also out of your mouth. 

To these preliminary remarks need be added only whatsis 
said under the special diseases, and some general cautions. 
The culture-room should be wiped up frequently with clean 
water. Generally, one's coat should be removed and his shoes 
cleaned before entering it. Fragments covered with spores 
of molds should never be taken into this chamber, nor should 



METHODS OF RESEARCH: TECHNIC OF ISOLATION 109 

any vegetable fragments be left there to mold later on. Select 
for isolation experiments the most recently diseased parts of 
the freshest material available. Further, because saprophytes 
often supplant parasites very speedily in diseased tissues and 
because even in the absence of these there may have been whole- 
sale death of the parasite owing to the acids or other by-prod- 
ucts it has liberated, thick sowings and other special devices 
are often necessary in order to isolate the cause of the disease. 

CARE OF CULTURES 

Room Temperatures. — When first made, the plates naturally 
must remain for a short time on a level shelf in the culture room, 
but as soon as solidified they must be removed to a suitable 
safe place for incubation, viz., to a clean dry cupboard, free 
from insects and protected from the light, at least from direct 
sunlight, or bright reflected light. If the laboratory is a 
clean one there should be little trouble from the larger vermin 
such as rats, mice, flies or cockroaches, but minute animals such 
as mites and small ants often live in the walls of buildings, be- 
yond reach, and may sometimes cause much annoyance in clean 
places. We have been troubled with both of these pests and 
now, as a routine practice, keep our plates and other cultures on 
shelves supported on cork legs resting in glass dishes containing 
mercuric chlorid dissolved in water, but after the water has 
evaporated more need not be added since the crystals uniformly 
distributed over the bottom of the plate are a sufficient barrier. 
The shelf, of course, should not anywhere touch the walls of the 
cupboard. This has proved quite effective. The pest of ants 
may be done away with by finding and destroying the nest, 
which is sometimes a long distance away. A good plan is to 
drive deep holes into the ground by means of a crow bar and 
fill these with kerosene. Record the temperature daily. 

Thermostats. — These are usually only for temporary use and 
the temperatures are to be shifted as needed, or rather some days 
in advance of the need, since often they are hard to regulate. 
We use the Roux metal-bar thermo-regulators. 



110 BACTERIAL DISEASES OF PLANTS 

Ice Boxes. — Stock cultures must be kept at low tempera- 
tures. They live much longer when cool and thus the labor of 
transfer, which is always considerable in any laboratory dealing 
with many organisms, is minimized. 

Our stock cultures are kept in ordinary refrigerators such 
as housewives use. This corresponds probably to the practice 
of many other laboratories but it is not the best way, since 
occasionally through carelessness on the part of the ice man or 
of servants the outlet becomes stopped up or the floor support- 
ing the ice becomes cracked and water drips down on some of 
the cultures, destroying them or at least contaminating them 
so that they must be plated out. A better way would be to 
keep stock cultures in a thick-walled roomy vault constantly 
supplied with cooled air. 

Ice Thermostats. — We make frequent use of the Paul Alt- 
man device containing ten small compartments. This, when 
the upper large chamber is filled morning and night with cracked 
ice, gives temperatures ranging, except during about four months 
of our year (June to September), from 0.5°C. to 20°C. These 
temperatures are not absolutely constant under our room con- 
ditions, but would be nearly so in a room having itself a fairly 
constant air temperature. By looking frequently and adding 
more ice if the temperature is observed to be rising, we are en- 
abled to make much use of it in determining lowest temperature 
at which seeds will germinate, microorganisms grow, etc. It is 
not, however, a perfect instrument. 

STUDY OF CULTURES 

All the various plant pathogenic bacteria should be grown 
on a great variety of media for discovering special characteristics, 
useful in identification, and also to determine the media best 
suited to their growth and longevity. These cultures should be 
examined frequently with the hand lens in a variety of lights, and 
often under the compound microscope, and should not be dis- 
carded for several weeks. Of course, checks should be held. 
To what I have said in other places, I would add here that 
colonies on agar and gelatin plates should be photographed in 



METHODS OF EESEARCH : STUDY OF CULTURES 111 

various lightings, enlarged ten times (that being recommended 
as a convenient standard magnification). This is so useful for 
purposes of comparison (Figs. 25, 114, 230, 254) that I have 
made it a routine practice in my laboratory for several years. 
In particular, oblique transmitted light will often reveal an inner 
colony-structure (Figs. 13, 37, 69, 275) not visible on the surface 
or by direct transmitted light. 

METHODS OF INOCULATION 

Needle Punctures. — I have made much greater use of the 
needle than of any other instrument in making inoculations. 
Its wounds are slight. It carries into the plant a minimum 
quantity of the organism to be tested and inoculations made in 
this way conform more nearly to natural methods of infection 
than do those produced by instruments making coarse wounds 
and introducing excessive numbers of bacteria. I am always 
suspicious of results that can be obtained only by drenching 
and swamping tissues with foreign organisms. 

Syringes. — ^Those injection syringes are to be preferred which 
are simple in construction, easily separable into a few parts for 
cleaning, and not liable to spirt out fluids around the piston. 
The piston and barrel should be of glass, the latter, of course, 
graduated, at least to tenths of cubic centimeters. They are 
seldom necessary in plant pathological work. 

Infection Cages. — Cages 2 feet deep by 2 feet high by 3 feet 
wide are most convenient. They should have a strong sash 
frame open at the bottom but set on all sides (including the top) 
with window glass, and the whole front should consist of two 
swing doors closing against each other in the center and closing 
at the bottom on a 2-inch baseboard. Such cages will hold eight 
8-inch pots. Each time before use they should be autoclaved. 
In absence of conveniences for this they should be wiped inside 
with mercuric chlorid w^ater a day in advance of use. Plants 
on which bacterial suspensions have been sprayed may be ex- 
posed to moist air in such cages 48 hours without harm. 

Soil Inoculations. — The earth may be sprayed or drenched 
with water suspensions of the bacterial cultures, or infected 
by burying in it recently diseased plants. At the same time 



112 BACTERIAL DISEASES OF PLANTS 

the roots of some of the plants should be broken. In many 
instances it is necessary to break them in order to obtain infec- 
tions (see Part III, Nos. IV and V). 

Checks should be held in the same soil, or if not, and espec- 
■ ially if the experiments are to be few, the earth should be auto- 
claved in advance and the plants watered with sterile (auto- 
claved) water. 

Infection by Means of Insects. — The common way is to 
confine the plants in insect cages (like the infection cages but 
with fine wire netting substituted for glass) into which the in- 
sects are introduced after they have been allowed to feed upon 
recentl}^ diseased leaves, stems, etc. Night-feeding insects should 
be infected and liberated at night, being removed next morning 
or earlier, especially if voracious. In case of trees, the insects 
may be confined to special branches by means of fine mosquito 
netting or surgeon's gauze. Under ground, the insects may be 
confined to the roots of special plants by wire netting. 

TIME AND PLACE OF INOCULATION 

In studying a particular disease, the student will, of course, 
seek to inoculate those parts of the plant which naturally de- 
velop the disease — roots, tubers, stems, leaves, flowers or fruits, 
as the case may be. He must remember, however, as he exam- 
ines the plant, that the infection he has under observation took 
place some days, weeks, or months ago when the diseased part 
was in an earlier stage of growth and in making inoculations must 
govern himself accordingly. 

In many instances, inoculations on very young, turgid, 
rapidly growing stems, leaves and fruits offer best prospect of 
success. Always some of the inoculations, whether by needle 
puncture or by spraying, should be on such incompletely devel- 
oped organs (see Part III, Nos. IV, VII, XII, and XIV). 

Full-grown leaves, stems and fruits are resistant to many 
diseases (bean. No. VIII, pear. No. XII, and olive, No. XIII). 
On the other hand in case of soft rots of roots (carrot) and tubers 
(potato) the full-grown organs are quite susceptible provided 
they have not become flabby (Fig. 167). Certain cankers may 
also be inoculated through a wide range of months. 



METHODS OF RESEARCH: CARE OF PLANTS 113 
CARE OF INOCULATED AND CONTROL PLANTS 

Every plant has its own peculiar demands for soil, water, 
light and heat. Some plants endure crowding and poor earth 
much better than others, but all prosper best when their needs 
are respected. The student should treat his plants with some 
consideration. He should water them regularly and sufficiently 
but not excessively; should keep them free from plant lice, red 
spiders, mealy-bug, white-fly and other pests and to this end 
should carefully avoid introducing infected plants into clean 
houses; should not over-crowd them, should be quick to see 
when they require shifting to larger pots ; and especially should 
not try to grow plants requiring a cool temperature in warm 
houses, or vice versa. The last is like trying to ride two horses 
going different ways. He cannot learn how to care for plants 
without being much with them, nor without this expert knowl- 
edge will he ever be more than a bungler in plant pathology. 
It is not enough to trust that ''the gardener will attend to all 
this," nor is it right to feel that this part of the labor is rather 
beneath the attention of an aspiring man of science. Nothing 
is too small or too menial to receive painstaking and minute 
attention if you wish to achieve a worthy success. Remember 
then that your plants should be looked after every day as to 
soil, air, light, heat, water, space to grow in, and freedom from 
insects, nematodes and fungous parasites. 

Mildewed or fungus spotted plants and all that are dwarfed 
by nematodes or defective in any way should be thrown away as 
soon as discovered and good ones substituted. Have others 
coming on, for this purpose. Do a little thinking in advance 
of actual needs! Neglected plants often become stunted or 
''pot bound," to use the gardeners' expression, and are then 
worthless. 

Seedlings often perish from damping-off fungi, especially if 
they are over-watered or the soil is poor. Plants require the 
same amount of water on scarcely any two days, and the student 
must learn as speedily as possible when to give and when to 
withhold water. On sunny and windy days they require 
a great deal, on still, cloudy daj's very little water or none what- 



114 BACTERIAL DISEASES OF PLANTS 

ever. Seedlings and cuttings require only a minimum quantity, 
but that they must have. A single excessive watering of seed- 
lings, or cuttings, may destroy your experiment. 

Plants seldom do well if transferred in full leaf to very 
different conditions as to light, heat and moisture, e.g., from 
the hothouse to the laboratory or to the garden or vice versa. 

For hothouse experiments great care should be taken to 
select good soil, i.e., that free from nematodes, ants, and para- 
sitic soil fungi, such as Rhizoctonia or Fusarium. Often a whole 
year's work is lost by reason of nematode-infested or fungus- 
infested soil. If you have any reason to suspect the soil, it 
must be steamed or baked, or drenched with formalin-water 
before it is used. Bordeaux mixture sprayed upon the earth 
will sometimes stop the activities of a damping-off fungus. 

Ants may be kept off benches by having runways all around 
them and their upright metal supports capped at about 2 feet 
from the ground by metal cups containing oil in which stand the 
short legs of the bench. Feed ants sweetened tartar emetic. 

PREPARATION OF SECTIONS 

Free-hand Sections. — Good free-hand sections are very 
useful in the preliminary examination. They can be made only 
by one who has enough gumption to sharpen a razor as often as 
it becomes dull, which is about every day. It is worth while 
even in a busy semester to learn how to do this. Sometimes 
a good-natured barber will show you how. The Torrey razor is 
excellent for section cutting but it is no longer on the market. 

Turgid tissues cut better than flabby ones, which latter 
sometimes recover turgor if thrown into water. The material 
to be cut should be held in a cleft of elder pith, never between 
corks, which dull razors. After a little experience very thin 
sections can be made of many things; these are usually more or 
less wedge-shaped, but if they are thin enough to see through 
that is all that is required, since one seldom mounts for perma- 
nent preservation anything but microtome sections. 

Microtome Sections. — Read what is said under Apparatus 
about microtomes. There are several methods of making 



METHODS OF RESEARCH: PREPARATION OF SECTIONS 115 

sections on the microtome. The tissues to be sHced may be 
frozen and cut on a special machine, and this is the quickest 
way and the best way for preliminary studies, but it is of no 
value for permanent sections of tissues containing bacteria, 
since they diffuse out too readily, or they may be embedded in 
celloidin or paraffin and cut on the ordinary microtome, which 
is the best way for permanent mounts. I will limit my account 
to paraffin, describing the way it is done in our laboratory. 
First the tissues are fixed. We select for this purpose, clean, 
perfectly fresh, characteristic bits which should not be large. 
Various fixing agents are used; we often use Carnoy's fixative 
(>i glacial acetic acid, ^i absolute alcohol). They will fix 
better in many cases if the air is pumped out of them as soon 
as they are put into the fixative. I make this an invariable 
rule. After 24 hours in this solution (12 hours is enough in 
some cases) the pieces are shifted into 90 or 95 per cent alcohol, 
the next day into a second alcohol, and finally from absolute 
alcohol into mixtures of alcohol and xylol, then into pure xylol, 
a second pure xylol, to remove all traces of the alcohol, then 
into xylxDl-paraffin, then paraffin plus xylol, and finally into 
pure melted paraffin, from which after some hours they are trans- 
ferred to another melted paraffin in which they are embedded. 
There is some choice in the melting point of paraffin to be used for 
the final embedding depending on the climate. In Washington 
we use during the summer Griibler's paraffin melting at 56° to 
58°C., and during the winter that melting at 52°C. The em- 
bedded pieces are trimmed and stuck to one end of a small rec- 
tangular white pine block by means of a hot needle and a little 
melted paraffin. The number of the specimen, corresponding 
to an account in a record book, is written on one or more sides 
of the pine block with a lead pencil. The wooden block is now 
clamped into the microtome. If the material is soft, ribbon 
sections are cut; if hard, slant-stroke sections are cut. If the 
knife is dull, or if the tissue is gritty or contains crystals, or is 
imperfectly embedded, the sections will be torn and worthless, 
or nearly so. Occasionally when the tissues are full of calcium 
oxalate crystals we have to be content with torn sections, but 
ordinarily the tissue should be reembedded and a sharper knife 



116 BACTERIAL DISEASES OF PLANTS 

selected. Of course, in the preliminary selection of material 
for microtome sections, the greatest care should be exercised 
to avoid that which is sandy or gritty. Dull knives and too 
soft paraffin also tend to give sections which are crowded to- 
gether endwise and this is undesirable. Always, thin, machine- 
made sections are more or less wrinkled, and the folds must be 
straightened out with gentle heat before the sections are glued to 
the slides; i.e., while they are floating on the water. This may 
be done, slide by slide over a low flame, or more conveniently on 
a metal table (Fig. 54) warmed slightly by means of an electric 
current, after which the slides bearing the sections are set on 
end to drain and dry. There are various ways of sticking sec- 
tions to slides. We now commonly use prepared egg-albumen, 
a little of which is rubbed over the surface of the slide with a 
clean finger before water is pipetted on to the slide to receive 
the paraffin sections. 

After two or three days, i.e., when the slides are thoroughly 
dry they are ready for staining, and it is best that they should 
be stained and covered soon, so that they will not accumulate 
dust. In no case, if the material is good, should the stained 
sections be left uncovered. 

STAINING METHODS 

Bacteria in the Plant. — -The paraffin of the properly dried 
sections is melted away by very gentle heat, just enough to render 
it fluid and no more, or they may be put into the xylol unwarmed, 
if one is not in a hurry, and this is the better way, whereupon 
the slides are put immediately into xylol, and often into a second 
xylol, to complete the removal of the paraffin. They are then 
graded through pure ethyl alcohols (95 per cent, usually 2 jars) 
to remove the xylol, graded from this pure alcohol into water, 
or alcohol and water to receive the stain. After this they are 
graded back into absolute alcohol to remove the water, and from 
this into pure xylol to remove every trace of alcohol. From 
pure (water-free) xylol they are mounted in Canada balsam 
or dammar balsam. The least water in the xylol will give a 
cloudy shde. 



METHODS OF RESEARCH: STAINING METHODS 117 

The proper degree of stain for sections should be determined 
by inspection under the microscope during the process of stain- 
ing. The sHdes must be shaken or jarred as Uttle as possible 
during these transfers, and, after staining, the shifting through 
the graded alcohols must be swift, so that the stain shall not be 
all removed. In my sections I prefer a good strong stain. 

Many of the slides put up in this laboratory and formerly 
very good are now nearly or quite worthless because they have 
faded. This has been due partly to use of aniline dyes which are 
fugitive even to diffused light, but more often I think to overwash- 
ing or to the presence of unsuspected traces of acid in the Canada 
balsam. If you would avoid much future disappointment use 
the utmost care in the selection of balsam for mounting pur- 
poses and do not overwash. Valuable sections which have 
faded may be restored by dissolving off the cover-glass in xylol 
and restaining. No invariable or general rule can be laid down 
for staining bacteria in situ since plants and parasites both 
vary in their reaction toward stains. Some bacteria also diffuse 
out of the sections vexatiously. Under the various heads in 
Part III, special stains are mentioned when such have been found 
particularly useful. 

Gram's stain varied by the substitution of amylic alcohol 
for ethyl alcohol (generally referred to later as amyl Gram) 
has been found to give a clean sharp picture of the bacteria in 
a number of tissues, e.g., olive tubercle, angular leaf -spot of 
cotton, pear blight. 

Carbol fuchsin may also be mentioned here as a widely 
applicable permanent bacterial stain. Its chief objection is a 
tendency to over-stain the tissues of certain plants, e.g., those 
of the mulberry. 

Two other very good stains are iron-haematoxylin and 
Flemming's triple stain. Nigrosin (soluble in water) also some- 
times gives good results. 

Long since (1894) the writer resorted to methyl violet, 
preceded by a bath in tannin water to reduce the excessive stain 
of the host tissues in stems of cucumber attacked by Bacillus 
tracheiphilus, and the bacterial masses in the vessels still hold 
their massive blue stain on a pale background (1915) but the 



118 BACTERIAL DISEASES OF PLANTS 

bacteria have held the stain less than some substance between 
them. Where only tissue differences are to be brought out, 
methyl green followed by acid fuchsin (see Part III, No. XIV) 
leaves little to be desired, at least in certain plants, e.g., crown 
gall on the Paris daisy, sunflower or house geranium, where the 
parenchymatic tissues become red or pink, and the lignified 
tissues blue. 

Staining Bacteria from Cultures. — A variety of stains are 
useful for staining smears and streaks from dilutions of pure 
cultures e.g., methylene blue. Gentian violet, basic fuchsin, 
carbol fuchsin^ amyl Gram. It is important to start with clean 
slides or covets, to dilute the culture so that the bacteria shall 
not be crowded, and so to stain and wash that the bacteria 
shall be sharply and deeply colored on a clean clear background. 
A momentary bath in alcohol following the stain is sometimes 
serviceable, or in acid alcohol if the organism is an acid fast 
organism, or in iodine followed by ethyl alcohol if it stains by 
Gram's method. 

Flagella Staining. — This is more troublesome than either of 
the preceding and will tax to the uttermost the ingenuity and 
capacity of the average student. Some never learn to do it, 
most are able to accomplish it with perseverance. A very few 
become experts and obtain beautiful preparations with com- 
parative ease. To succeed, the culture must be suitable. One 
seldom obtains good preparations from cultures that are not 
actively motile. Some species are much more obdurate than 
others. Attempts should be made as a rule only from very young 
agar-streak cultures (6-24 hours) and not then unless an inspec- 
tion of the organism on the margin of a hanging drop shows it to 
be actively motile. Sometimes better success may be had by 
transfers from bouillon to agar than from agar to agar, or by 
flooding a young agar culture with 15-20 cc. of distilled water 
and then taking motile organisms from the top part of the fluid. 

The slides or covers used must be absolutely clean and of 
good quality. The mordanting fluid should be fresh. The 
bacterial film should be evenly spread and the individual bacteria 
widely separated but not too widely. The covers maybe lightly 
flamed before staining but this is not always necessary. Very 



METHODS OF RESEARCH: FLAGELLA STAINING 119 

important is the right length of exposure to the mordant and 
then the right length of exposure to the stain Only some such 
very general directions can be given because a method that will 
succeed with one organism often ;ails entirely with another The 
student should consult the various text books and then go ahead. 
We have had excellent results on a variety of organisms using 
half a dozen different staining methods. 

Special Stains. — For method of staining spores and capsules 
consult the various text books. The opposition to the use of mor- 
phological differences for classifying bacteria has come largely, 
I believe, from persons who are not naturalists and who have 
had indifferent success in staining bacteria. 

CARE OF SPECIMENS 

In many institutions there exists a discreditable lack of 
thoroughness in the preservation of interesting specimens, and 
those persons who throw away good pathological material are 
usually the very ones who have described it badly to begin with, 
so that between an imperfect description and entire absence of 
the materials on which the description was founded, the system- 
atist can only guess what was the real state of the case. Some- 
times when a laboratory changes heads the collections of the 
first man are destroyed by the second. I have known one shock- 
ing case of this kind. The student should remember that 
whatever is worth describing is also worth preserving. The col- 
lections of a pathological laboratory, if well made, become of 
greater and greater importance as time passes. They should 
include the following groups of material. 

Herbarium Specimens. — These are to be fastened on sheets 
of stiff white paper of the standard herbarium size, and properly 
labeled, one sheet being devoted to each parasite, but the more 
specimens of it the better, especially if they are from different 
localities. 

Coarse Dried Material. — Limbs, trunks, and other material 
too cumbrous for the herbarium sheets should be properly 
ticketed and stored in tight boxes or in glass cases. This 
material and the herbarium sheets must be examined frequently 



120 BACTERIAL DISEASES OF PLANTS 

for insect depredations and treated for the same like other 
herbarium material. 

Alcoholic Material. — Quantities of this should be preserved 
for two purposes: a, class use; h, permanent records and ex- 
hibition purposes. The former must be fixed in Carnoy's 
fluid or some other suitable fixative before transfer to the 
alcohol. Flat (parallel-walled) jars may be used for the ex- 
hibition collection, which may be set off by a background of 
milk-white glass placed inside the jars, though this is not abso- 
lutely necessary. Any alcoholic collection must be examined 
once a year and leaking jars refilled and resealed. 

Pathological material may be preserved, with the green color 
of leaves and stems retained, by boiling the specimens for about 
5 minutes, more or less (1 to 10 minutes) "in 80 parts of distilled 
water to which has been added 20 parts of glacial acetic acid 
saturated with copper acetate. The treated specimens should 
be rinsed in water and placed permanently in water containing 
5 per cent formalin, or in localities not subject to freezing they 
may be kept in sulphurous acid water (Gino Pollaci's method). 

Microscopic Preparations. — These may be laid in flat trays 
or in grooved wooden boxes. The writer uses the common 
Pillsbury boxes which hold each 25 slides. These slides have a 
gummed label on one or both ends bearing the block number and 
other necessary data, i.e., object, date, kind of stain, etc. These 
should be kept in the dark. The slide boxes in my laboratory 
are filed either under the name of the parasite or serially. 

PREPARATION OF ILLUSTRATIONS 

Photographs. — The general excellence of photographic ap- 
paratus in recent years, and the slight cost of plates and films, 
render this method of making records extremely serviceable. 
The student who is planning a career in science should determine 
from the start to be master of all the common methods of photog- 
raphy. In every locality, nearly, there is some one who can 
teach him the rudiments, and even if not, good journals and 
books on the subject are now numerous and inexpensive. There 
is, therefore, no excuse for neglect of this interesting and im- 



METHODS OF RESEARCH! PREPARATION OF LLUSTRATIONS 121 

portant aid to research. How important it is, will be evident 
at once if we reflect that photography enables us to fix the thou- 
sand and one fleeting phenomena of nature in a permanent record 
with which to refresh our memory and from which to draw, in 
a way quite beyond the power of former generations, convincing 
illustrations for papers, books, and lectures Not infrequently 
the study of a good negative reveals details overlooked on the 
object itself. This is most important, of course, in astronomy, 
but it also has its uses in pathology. 

Remember that one good picture is better than half a dozen 
pages of text in hammering home your argument and that if 
you fail to convince your readers and hearers, then your work 
i s in vain. Therefore, do not spare good illustrations . 

The following fragmentary observations are drawn from our 
own procedure and may be of service in smoothing the way for 
the beginner. 

Good lenses, light-proof bellows, and suitable dry plates 
are essential to the making of good photographs. The student 
should read what is said on this subject under Apparatus. 

Pathological subjects usually show strong contrasts and to 
get similar contrasts on the negative often taxes the skill of 
the expert. It took the writer and James F. Brewer two days 
to get the result shown in Fig. 281. 

Development should be carried on in the dark as much'as 
possible. When light is allowed to reach the developing plate 
it should be only of a minimum brightness (very dull red) and 
for only very brief periods, especially during early stages of 
development, and at all stages of development in case ot plates 
corrected for the red end of the spectrum 

For ordinary work and for certain color contrasts such as 
white and black or red and blue, Seed's rapid No. 30 Gilt 
Edge dry plates may be used. These are not sensitive to red 
light, therefore contrasting reds, yellows and greens wil come 
out dark on the prints and often be disappointingly alike, while 
the overexposed blue and violet parts will be pale or white on 
the prints. Consequently, for many subjects where color con- 
trasts are desired, Cramer's Iso medium or Iso slow plates may 
be used. Better still for this purpose are Wratten and Wain- 



122 BACTERIAL DISEASES OF PLANTS 

Wright's Panchromatic plates, which give nearly perfect color 
values, and for this reason are so sensitive to red light that they 
should be developed from start to finish in the dark, recommen- 
dations to the contrary notwithstanding. Even with the best 
plates it is sometimes impossible to get the contrast of nature 
unless color screens are used. See Fig. 281. Here a bluish 
black stripe on a pale green ground, perfectly distinct to the 
naked eye, defied all attempts at photographing until the ex- 
posure was made through a yellow screen. See also Fig. 239 of 
cotton leaves and bracts and Fig. 47. 

As a developer, rodinol (sometimes called cytol), which is 
ready for use on dilution with water, leaves little to be desired. 
Many developers are poisonous and iritating to the skin. The 
hands, therefore, should be kept out of them as much as possible 
and always washed immediately after they have been dipped 
into the tray. 

Prints for filing, or for use of the photo-engraver should be 
on paper that does not curl. Films or papers that curl are 
a source of endless vexation. We now employ two styles of 
Eastman's Velox paper, viz., Special Glossy Velox paper for 
hard (contrasty) negatives and Regular Glossy Velox paper for 
soft negatives. In printing, dense negatives may be exposed to 
daylight for a few seconds (10 to 20, at some distance from the 
window), but thin negatives must be exposed to a weaker light. 

Reproductions from photographs are usually made on copper 
by the half-tone process. Half-tones illustrative of patholog- 
ical appearances (details) should be made with a screen not 
coarser than 150 meshes to the inch, and in many subjects im- 
portant features will be lost if the screen is not finer, i.e., 175 
meshes to the inch. On good paper, with a good pressman (and 
this is all important), there is no difficulty in getting a clear 
impression from a well-made half-tone having 175 meshes per 
inch, only the paper must be suitable, must be backed up properly 
and printed carefully with just the proper amount of good ink. 
Many of the smudges passing for illustrations in current publi- 
cations are a disgrace both to the scientific man and to the printer. 
I provide against loss of important small details through un- 
avoidable defects in the half-tone, by enlargements to twice 



METHODS OF RESEARCH: PREPARATION OF ILLUSTRATIONS 123 

natural size with the ordinary lenses and an extra long bellows, 
or by means of planar enlargements. These latter enable one 
to show distinctly even the minutest color changes, or form 
changes, on leaves, stems, fruits, or other organs. 

Frequently when a very good photograph has been furnished 
the engraver, he ruins the plate he has made from it by over- 
etching. In such cases he should be compelled to make another. 
But the best half-tone ever made can be ruined by a second rate 
pressman. For an example of a good plate ruined by a slovenly 
pressman see ''Bacteria in Relation to Plant Diseases," Vol. I, 
page 178, and for a properly printed half-tone from the same 
negative, see Fig. 67 in this book. 

Enlargement to twice natural size requires the ground glass 
(focusing plate) to be from the lens a distance equal to three 
times the equivalent focus of the lens, i.e., the enlargement of an 
object to twice natural size, using a lens of 93^ inch focal length, 
requires the placing of the object 14i^ inches in front of the 
lens and the bellows extended to about 28 inches. 

Photomicrographs. — Success with the large horizontal photo- 
micrographic apparatus formerly much used by the writer 
depends on keeping a few things constantly in mind: (1) the 
source of light must be reasonably constant ; (2) the rays of light 
must pass in a straight line from the luminary to the ground glass, 
i.e., every piece of the apparatus must be centered; (3) a ray filter 
should be used ; (4) if the bellows is elongated to twice its former 
length the exposure must be quadrupled, and vice versa; (5) 
if high power lenses are substituted for low power ones the ex- 
posure must be lengthened (one comes after a time to judge 
very correctly of the exposure by the amount of light on the 
ground glass, but the beginner will save much time and vexation 
by using an exposure shutter, and in its absence by drawing 
the ordinary slide }.i, then ^i, ^i, and finally entirely, and judg- 
ing by development with a normal developer which of the various 
exposures, or between or beyond which, will give the best re- 
sults) ; (6) the time required for unstained and thin-stained slides 
is very much less than that for those heavily stained, and in 
exposing thick or dark-stained slides the time must be long enough 



124 BACTEEIAL DISEASES OF PLANTS 

to allow the light to penetrate the thickest parts; (7) no specific 
rules can be given as to length of exposure. It varies enor- 
mously with the subject, objective, eye-piece, bellows length, 
size of diaphragm, sensitiveness of dry plate, kind of screen and 
source of hght, and if sunshine is used, state of the sky, latitude, 
time of day and time of year. In Washington, using planar 
lenses with short bellows length and bright sun light, it is gen- 
erally only fractions of a second (sometimes as short as j^foo 
second). With high powers, long bellows, small stop and dark 
subjects it is often five minutes or more. Sometimes it is very 
much longer if the source of light is feeble. Using the light of 
the open sky, Cramer's Iso slow plates, and a densely stained 
slide, I have sometimes exposed the plate for an hour, but this, 
of course, is exceptional, although under such conditions 10 to 
20 or even 30 minute exposures are common for magnifications 
of X 1000. Toward sunset the actinic effect of the light falls off 
very rapidly and the exposure must be correspondingly lengthened, 
30 minutes to an hour being not unusual. 

If the object is a small one in the center of an otherwise 
well-lighted field, the exposure should be much shorter, naturally, 
than when there are deeply stained objects in many parts of the 
field. 

The beginner will do well to confine himself to the simple 
upright camera. If he avoids direct sunlight, focusing on the 
diffuse light of the sky, he will have more leeway in his exposures 
and will not require a shutter. The exposures with such an 
apparatus are very much lengthened, even on clear days, as 
compared with arc light or heliostat light, but this is no serious 
difficulty. With such an apparatus, using Cramer's Iso-chro- 
matic slow plates, a Zeiss 2-mm. apochromatic oil-immersion 
lens, a No. 4 compensating ocular, and a magnification of X 1000, 
the time of exposure here in Washington on a clear summer day 
between 9 a.m. and 3 p.m. varies, with the nature of the section, 
and the stop, from one minute to 20 minutes or more; with all 
else the same, but using a Zeiss 16-mm. apochromatic objec- 
tive, the time varies usually from five seconds to 10 minutes ac- 
cording to the subject and the stop. A little experience will 
enable the painstaking student to obtain good pictures, especially 



METHODS OF RESEARCH: PHOTOMICROGRAPHS 125 

with low powers. The field must be flat, must be uniformly 
lighted, and the proper focus must be obtained, which focus is 
not exactly that of the eyepiece, but must be secured by use of a 
hand lens on the ground glass, or preferably on clear glass 
substituted for the ground glass. I first focus with the eye- 
piece, then attach the bellows and focus on the ground glass 
under a thick dark cloth (two folds), at which time I judge of 
the uniformity of lighting and center my object, then I sub- 
stitute the plain glass for the ground glass and re-focus, using a 
hand lens. Last of all, I stop-down, always considerably, and 
make the exposure. In all this I am considering only apochro- 
matic objectives. With some of the older Zeiss achromatic 
objectives I found it impossible to get a focus upon the ground 
glass that would photograph. In this case I determined experi- 
mentally by a series of photographs made at slightly different 
levels the amount of the error and thereafter obtained a sharp 
focus on the ground glass, turned it out the required amount, and 
then made my exposure. 

Photomicrographs require a longer development than ordi- 
nary photographs since in the latter we aim at a pleasing grada- 
tion of tints whereas we usually have in the stained slides sharp 
contrasts and wish to retain these contrasts on the negative. 
We use hydroquinon developer (double the amount of hydro- 
quinon of Cramer's formula with the metol left out) and I 
develop until the image is well through on the back. This 
occurs in a fully exposed plate in seven minutes, in a slightly under- 
exposed one in 15 to 25 minutes. If I wish very marked con- 
trasts such as deeply stained bacteria on a white background 
(Fig. 187 for example) I under-expose so that the image comes 
up in five to seven minutes rather than in two minutes, and I 
develop a long time (30 or 40 minutes), with a hydrochinon 
contrast developer, so as to have a very dense background. 

The plates are fixed in the green (chrome alum) acid fixing 
bath where they should remain at least 20 minutes, and much 
longer will not hurt them. We prepare the bath, only when we 
are ready to use it, from standard stock solutions which keep 
indefinitely separate, adding to four tumblers of the filtered 
saturated water solution of sodium hyposulphite (water 128 



126 BACTERIAL DISEASES OF PLANTS 

oz., hypo. 32 oz.), slowly stirring in, with a glass rod one tumbler 
of the green fluid (water 32 oz., dry sodium sulphite 3 oz., c.p. 
sulphuric acid J2 oz., powdered chrome alum 2 oz., dissolved 
in the order named). 

Lumiere Plates. — Consult a booklet on ''Color Photography 
With Autochrom Plates." R. J. Fitzsimons, 75 Fifth Avenue, 
New York, Agents for Lumiere Jougla Products. 

Planars. — The use of planar lenses has greatly simplified 
the work of the pathologist. By means of magnifications rang- 
ing from X5 to X25 they enable him to show every detail of a 
surface not too irregular, and the whole of sections much too 
large for the ordinary microscope. I make most use of mag- 
nifications ranging from X5 to Xl5 and prize these lenses 
very highly. Their only defect, if such it be, is that the field 
must be quite flat, if the image is to be uniformly sharp. There 
is, however, a little penetration and to make the most of this 
we usually focus with the diaphragm wide open and then stop 
down as much as possible. The final focusing should be on 
clear glass, using a hand lens magnifying about X6. What 
can be done with such lenses is shown in some of the illustra- 
tions in this book. Figure 72 was one of the hardest to make 
because the section is thin and now shows scarcely any stain 
except in the bacterial masses, and because it was made on the 
upright camera without a color screen. 

Drawings. — At one time the pendulum of scientific senti- 
ment swung away from drawings toward photographs, because 
it was said the drawing represents only the ideas of the artist 
whereas the photograph cannot lie, but now it has come back 
again, since it has been recognized that the latter statement is 
not strictly true (Fig. 2106, c), and that the former objection 
lies chiefly against artists who are not naturalists. 

For many purposes, a good drawing is better than a good 
photograph, but there is no reason why the scientific man should 
not use both. I have heard many students say: "I can never 
learn to draw," but with few exceptions such is not the case. 
Every student should be encouraged to use pen and pencil 
every day to illustrate what he sees. The more he does it under 
proper guidance the more he will see. Certainly he has not 



METHODS OF RESEARCH: DRAWINGS 127 

seen an object to much profit if he cannot make at least a 
sketch of it, and this, however rude, is better than nothing, and 
helps just that much toward his intellectual development, 
which, after all, is the end of most of his studies. 

Drawings may be made with lead pencil, pen and ink, or 
the brush. 

Pencil drawings well made, are very attractive, and if there 
is not a question of expense they may be used to illustrate 
scientific papers by means of lithography, but this process is 
much more expensive than line engraving (zinc process) or 
half-tone plates, and is now seldom used by publishers except 
for expensive works. Drawings designed to illustrate books 
and papers should, therefore, be made, as a rule, with India ink, 
by use of pen or brush. The latter, known as wash-drawings, 
can only be half-toned. The former are generally reproduced 
by the zinc process. Zinc cuts are less expensive than the cop- 
per half-tones, print well, and are very attractive if well made. 
To get good results the ink must be black and non-soluble in water, 
the lines or dots must be distinct in all parts and must be prop- 
erly spaced, so that when the drawing is reduced, none of them 
will fade out in places, owing to insufficient or pale ink, or run 
together into blotches because not properly spaced. The 
student should have a clear idea in advance of where he intends to 
introduce his lights and shades so as to produce an attractive 
picture, and how any part will appear when it is reduced one-half 
or two-thirds. For this reason he should examine his drawing 
from time to time under a reducing glass, and when it is done 
not allow it to be reduced more than it will stand. I recall 
seeing many years ago a hundred beautiful pen-and-ink drawings 
made by one scientific man for the use of another. They were 
drawn and marked to be reduced, according to my recollection, 
just one-half and would have stood that amount of reduction 
perfectly, but for reasons of economy they were reduced twice 
the proper amount with disagreeable blotchy results, satisfactory 
neither to the artist nor to the author. Whenever in doubt 
the finished drawing should be photographed at the pi'oposed 
reduction. This is a sure way of deciding how much re- 
duction any drawing will stand. A finished drawing represents 



128 BACTERIAL DISEASES OF PLANTS 

much labor and should be carefully protected (covered) until 
engraved. 

Very much can be done by line work alone, but the finest 
results, especially where details are important, can be achieved 
only by making the drawing partly or wholly of large and small 
dots (stipple), placed close together and wide apart as the shading 
or other features require. As such drawing is rather hard 
on the eyes it should generally be done under a drawing glass 
magnifying two or three times, and always should be drawn 
larger than the desired reproduction so that small inequalities 
in the hand work may disappear or blend into a general smooth 
whole when it is reduced one-half or two-thirds. Great care 
must be taken to lay the ink on properly so that the reduction 
shall look uniform and attractive. Such drawings may be made 
direct from the microscope or inked over a pencil sketch, or 
may be laid over a pale silver print on salted paper, or a blue- 
print, or a print on bromid paper, the remaining photographic 
details of which are then bleached out in a bath (the importance 
of the water-insoluble India ink is now apparent) after which the 
sheet is dried and retouched here and there, as necessary, to 
fill in places which were overlooked. 

The bleaching baths are as follows : 

1. To bleach silver-salted paper: use a weak solution of 
mercuric chlorid in alcohol (poison). 

2. To bleach blueprints: use a dilute solution of potassium 
carbonate and potassium hydrate after which the print should 
be flowed with dilute hydrochloric acid and washed. 

3. To bleach bromide prints: 

a. Use Thiocarbamide 120 grains. 

Nitric acid 2 fluid drams. 

Water 10 ounces. 

Wash and dry. Safe but slow. 

or h. Use a saturated solution of iodine in 

alcohol 2 drams. 

Saturated solution of potassium 

cyanide in water 3 drams. 

By adding water the action becomes slower. 
Note. — This solution is very poisonous. 



METHODS OF RESEARCH: DRAWINGS 129 

For ordinary pen-and-ink or wash-drawings we generally use 
Reynolds' English Bristol board and Higgins' water-proof 
India ink. 

The first essential of a good drawing, and the sine qua non, 
is fidelity, but one may have kept that and yet the drawing may 
be unattractive, i.e., all black or all pale. Generally I prefer 
to indicate certain tissues by shading them and I select these 
arbitrarily in such a way as to give to the drawing a varied and 
pleasing effect. This is gratis! 

When letters or figures are introduced into photographs or 
drawings the intended reduction should always be taken into ac- 
count, i.e., the letters and figures must be large enough so that 
they may not be lost or obscured when reduced, since much 
time is sometimes wasted in trying to read an author's references. 
For the same reason, when drawings numbered serially are cut 
apart and rearranged on a plate, as sometimes happens in an 
effort to economize space, they should be renumbered serially 
from left to right and top to bottom. This is laborious to the 
author, but the labor involved is as nothing compared to the 
burden the neglect of it throws upon a thousand readers, par- 
ticularly if the figures are numerous and the lettering indistinct. 

Paintings. — Water-color drawings are often indispensable, 
since neither photographs nor ordinary pen or pencil drawings 
can convey color effects satisfactorily. A faint pencil outline 
is usually sketched in and then the proper colors are flowed 
over the surface with a swift sure touch. The technic is very 
different from that of painting in oil, which may be worked over 
and over, since in a water-color the right pigments must be laid 
on swiftly once for all, the drawing admitting afterward only 
of a minimum amount of correction. It is nevertheless greatly 
to be preferred to oil, because it gives a smoother surface and 
brighter colors. Not many students will be able to make their 
own water-color drawings. When such are necessary some one 
can usually be found to do it. Since such persons are usually 
untrained, scientifically, in my own case I have always found it 
necessary to superintend the process, suggesting now and then 
the colors to be sought for and the details to be emphasized. 
In this way I have succeeded quite well with various artists 



130 BACTERIAL DISEASES OF PLANTS 

having none of the naturaUst's outlook, and unable in my judg- 
ment to do it alone. The trouble generally is that the artist 
sees color rather than structure and strives for general effects 
by omission of details, whereas the scientific man desires his 
colors to be laid on carefully over definite structures. This is 
not a criticism of the artist as such, but only of the artist turned 
naturalist. The ends aimed at are different and each man is 
right in his own field. 

To see examples of the various kinds of drawings and what 
I mean by the above remarks, the student may consult "Bac- 
teria in Relation to Plant Diseases," Vol. I, Figs. 1 (drawn from 
a photomicrograph), 2 and 3 (drawn directly from the micro- 
scope), 4 (drawn from a photomicrograph and reduced), 39, 
63, 64, and 86 (good line drawings), 132 (rough but effective line 
drawing) . 

Ibid., Vol. II, Figs. 4 (drawn unstained), 12, 15, 16, 17 (drawn 
from stained sections directly from the microscope), 69 (drawn 
directly from the microscope), 80 (a good wash-drawing re- 
duced too much, compare with photomicrograph of same sub- 
ject in this book, Fig. 72), 102 (inked over an enlargement from 
a photomicrograph), 118 (inked over a photomicrograph and 
then much reduced), 137 (drawn directly from the microscope). 

Ibid., Vol. Ill, Figs. 7 (drawn from a stained slide), 16 (drawn 
from a broken negative), 19, 20, and 21 (drawn large from 
stained slides and much reduced — contrast 20 and 21 for 
different methods of shading), 72 and 75 (drawn directly from 
stained slides), 84 (drawn from the slide and much reduced), 
86 (drawn from a photomicrograph), 105 (wash-drawing much 
reduced), 108 (drawn from the slide but slightly diagrammatic). 

CARD CATALOGUES AND SYSTEMS OF FILING 

Every successful student of the natural sciences must of 
necessity read widely and, since memory in civilized man is an 
uncertain way of retaining knowledge, records of some sort are 
absolutely necessary. Loose sheets or a pocket notebook serve 
the ordinary man, but as knowledge widens, as many subjects 
come under observation, and as scientific materials accumulate, 
orderly catalogues and systems of filing become indispensable. 



METHODS OF RESEARCH: CATALOGING AND FILING 131 

Standard library cards are recommended for the literature 
catalogues. These should be of good size so as to admit of a 
short summary of contents, if thought desirable. They may be 
typewritten or else written in black ink and in a handwrit- 
ing plain as print. For various other catalogues smaller cards 
are sufficient. We keep an index of negatives, of paraffin- 
embedded material, of culture media, of stock cultures, etc. 
Our stained sections are filed in Pillsbury boxes, standing on end 
in deep drawers. The paraffin block number is written on the 
end of each box, and sections of all the hosts attacked by a para- 
site are filed together under the name of that parasite. 

Our negatives are placed in strong paper negative bags and 
filed in deep drawers. They are arranged alphabetically ac- 
cording to the species name of the parasites, with lettered guide 
boards separating the photographs relating to the different 
parasites, and the photographs of each subject are numbered 
serially on the face of the envelopes in red ink. 

Each negative bag has all necessary data written on it, but 
this is not enough since various persons handle these negatives 
and it is extremely easy to get the negative covers misplaced, 
so that often after years have elapsed one is in great uncertainty 
as to which belongs to which. I make it an invariable rule, 
therefore, to write all necessary data on the margin of the nega- 
tive itself, carefully selecting a place where the writing will not 
interfere with the printing. In fact, I consider a negative with- 
out such a record on it as worthless, no matter how good it is in 
all other respects, because it is like a natural history specimen 
whose locality has been lost. 

Negatives should never be filed without covers since if they 
are on glass they are certain to become scratched or broken. 
It gives me the shivers to see the way in which some scientific 
men treat negatives. 



PART III 

SYNOPSIS OF SELECTED DISEASES 
I. THE CUCURBIT WILT 

Type. — This is a wide-spread, typically vascular, wound- 
infection, wilt disease (Figs. 62, 63, 64), transmitted by insects 
(Figs. 65, 66, 67). The plants wilt suddenly without any visible 
cause, and on cross-sections of stems of such plants there is a 
viscid, white bacterial ooze from the vascular bundles (xylem 
part). The disease is first visible on the leaves locally in the 
form of dull green, rapidly extending, flabby patches. Later all 
the leaves wilt and shrivel. It is not known outside of Cucurbit- 
aceae and some members of this family are not subject. It is 
believed to be non-tropical in its distribution. Its range toward 
the equator is unknown. Rand has recently discovered it in 
the United States as far south as Florida. It occurs in Europe, 
South Africa, and Japan. In hothouses the disease occurs not 
infrequently in the winter season, often destructively, and here 
again insects are the carriers. 

Cause. — It is due to Bacillus tracheiphilus EFS.^ This is a 

1 Note. — The generic names in this book are used by me in the following way : 

Bacillus: A rod-shaped schizomycete, motile by means of peritrichiate flagella. 

Bacterium: A rod-shaped schizomycete, motile either by means of one polar 
flagellum or several such flagella. 

Aplanobacter: A rod-shaped, non-flagellate, non-motile schizomycete. 

For a discussion of the principles involved in this nomenclature, see "Nomen- 
clature and Classifications" in "Bacteria in Relation to Plant Diseases," Vol. I. 

The peptone referred to in this book is Witte's Peptonum siccum. The 
agar is Kahlbaum's Pulverized Agar Flour. The gelatine is Nelson's Shredded 
Photographic Gelatine, No. 1. All titrations referred to were made with N/20 
NaOH, or N/20 HCl, using phenolphthalein as indicator, unless otherwise 
specified, the merest trace of the pink color being taken as the zero point. 
Bouillon means 1 per cent peptone-bouillon unless otherwise indicated. Milk 
means the fresh cream -free product. As already stated in Part II, the color 
changes in most cases have been compared with Ridgeway's Color Scale {Ri 
or R2). Sometimes, however, there is no color in Ridgeway's system that fits, 
and in such cases I have used common terms. 

132 



THE CUCURBIT WILT: CAUSE 



133 




Fig. 62. — Cucumber wilt due to Bacillus tracheiphilus. A natural infection. 
Photographed by the writer in 1893. 




Fig. 63. — Squash wilt due to Bacillus tracheiphilus. A natural infection. (After 

Clinton.) 




Fig. 64. — Muskmelon wilt due to Bacillus tracheiphilus. Result of a pure culture 
inoculation made April 15, 1896. 



134 



BACTERIAL DISEASES OF PLANTS 









W^^ 



Fig. 65. — Portion of a cucumber leaf wilted by Bacilluti tmchtdpJtilas (which 
was abundant in many of the smaller veins) and then greedily gnawed by the 
striped cucumber beetle {Diabrotica vittata). Such beetles are then infectious to 
other plants. 




Fici. 66. — A healthy cucumber leaf slightly gnawed by an infected striped beetle 
and developing the bacterial wilt around the bitten places. From a field near 
Washington, 1893. 



THE CUCURBIT WILT! CAUSE 



135 



viscid, capsulate, motile, white, 
non-sporiferous, slow-growing, non- 
liquefying, non-milk-curdling, non- 
nitrate-reducing, non-gas-forming, 
aerobic and facultative anaerobic 
(acid-producing), rod-shaped, peri- 
trichiate schizomycete (Fig. 68), 
forming on the surface of agar- 
poured plates, internally reticulated 
(Figs. 69, 70), small, circular 
smooth, wet-shining (Fig. 71), 
colonies. It is easily killed by 
heat, by dry air and by weak 
acids. It must be transferred very 
frequently on culture-media. It 
may be kept alive longest in milk 
or in sugared peptone water over 
calcium carbonate. It does not lose 
virulence quickly. There are at 
least two strains, to one of which 
(f. cucumis EFS.) the squash is im- 
mune. 

Technic. — If the organism to be 
used in the inoculations must be 
isolated from old stems, wash the 
stem thoroughly in clean tap water, 
select a middle part, roll around 
quickly two or three times in a 






Fi(i. 67. — Secondary (general) bacterial 
wilt of a cucumber vine as the result of bites 
of the striped beetle {Diabrotica vitiata) after 
it had fed on a bacterial culture. These 
bites, few in number, were on the lower leaves 
and resembled those shown in Fig. 66. The 
infectious material was obtained by the 
beetles from leaves of other cucumbers where 
I had placed it a few hours before in making some needle-prick inoculations. 
Beetle-bites both on my inoculated leaves and on those of this plant were dis- 
covered next morning and the disease was predicted. It developed around the 
bitten places in the usual time and progressed just as on the pricked plants. 




136 BACTERIAL DISEASES OF PLANTS 

Bunsen flame, or slowly in the flame of an alcohol lamp, so 
as to singe the surface without cooking the interior. Place 
the flamed part on a sterile surface (a strip of glass or a clean 
board passed repeatedly through the Bunsen flame, or the in- 
side of a baked Petri dish) and cut crosswise with a red-hot 
knife. If you have any doubt as to the sterility of the cut 
surface, i.e., if you have accidentally touched it with your 



tv#.": '^^' -c 



*.^. 



'4 



1 



:r^ 







Fig. 68. Fig. 69. 

Fig. 68. — Flagellate rods of Bacillus tracheiphilus stained by van Ermengem's 
silver nitrate method: a, b, stained and photographed by the writer in 1904. The 
oi'iginal negatives are X 1000, but the picture here shown has been made X 2000 
to bring out the flagella more distinctly; c, stained by Mary Katherine Bryan, in 
1915, and photographed by the writer. X 1000. 

Fig. 69. — Agar poured plate smooth surface colony of Bacillus tracheiphilus 
enlarged 25 times and photographed by transmitted oblique light to show internal 
markings; 5th day, 1919. 

fingers or have dropped it, press the hissing hot knife firmly once 
or twice for a moment or two on the cut end of the stem. Sur- 
face organisms are thus excluded. Then squeeze the stem, 
forcing bacteria from the unheated part to the cut surface for 
your cultures. 

The slime of this organism within the vessels of the stem 
is usually so viscid that plate cultures made from it frequently 
miscarry. Along with this method, therefore, try a second, i.e., 



THE CUCURBIT WILT: TECHNIC OF ISOLATION 137 

at the time the plates are poured inoculate a tube of bouillon 
or of peptone water by squeezing into it a drop of the viscid 
fluid, let it stand 18 to 24 hours, and then pour from it a second 
set of plates, inoculating them very sparingly from the original 
tube, if it is well clouded, and rather copiously if it is clear. 
Inoculate also from dilutions of the same. Save all tubes, or 
at least the dilutions until results are known. The diseased 
stems must also be retained until results are known, unless 
others are easily procurable. They may be kept in the ice box. 

For inoculation purposes use cucumber, muskmelon, squash. 
These should be planted in a warm place 2 months before they 
will be needed, should be potted off early, and shifted to larger 
pots frequently to keep them growing rapidly. Watermelons 
may be inoculated for contrast. These cucurbits require houses 
having a day temperature varying from 65° to 75°F., and a night 
temperature about 15°F. lower. Avoid chilling the plants as 
then they are very subject to mildew. If any mildewed plants 
appear they should be removed from the house immediately. 

Inoculate by needle-pricks (10 to 20 to make sure, but on 
some a lesser number for comparison), using young (2- to 6-day) 
agar-streak, potato, peptone, or bouillon cultures. Prick the 
blades of well-developed vigorous leaves. Make check pricks 
on other leaves of the same plant, or upon the same leaf on the 
opposite side. In both cases group the pricks. Examine the in- 
oculated leaves frequently from the second day on. Do not 
over-water so as to complicate by root-rot. Do not tear the 
leaves in making inoculations. 

Determine 

For the organism. Morphology. — In various media: size 
in microns, form, aggregation of elements if any, i.e., chains, 
filaments, pseudozoogloeae, etc., motility on margin of hanging 
drop (using a high-power dry lens or an oil immersion lens, with 
a long working distance) ; absence of spores (heat, spore stains) ; 
presence and distribution of flagella (van Ermengem's stain); 
stain one of the viscid, cobwebby threads on a clean slide 
using carbol fuchsin and study under a high magnification. 
Try capsule stains. Gram's stain. Do involution forms occur? 



138 BACTERIAL DISEASES OF PLANTS 

Cultural Characters. — ^Growth on thin-sown agar-poured 
plates — internal structure of surface colonies; growth in gelatin 
plates; stabs and streaks on agar; do. gelatin. Appearance on 
steamed potato : this should be wet-glistening, and almost ex- 
actly the color of the white surface of the potato. A non-para- 




FiG. 70. — Internal markings of agar surface colonies of BaciUua tracheiphilus: 
a, Isolated from a wilting plant bitten by a striped beetle (1915); h, c, Rand's 
No. 230 checked up by the writer on cucumber (1918); d, Rand's No. 183 plated 
in 1919, also checked up on cucumber. All photographed by oblique transmitted 
light. X 10. 

sitic coccus follower answering to this description on steamed 
potato may be distinguished by the fact that it reddens litmus 
milk. Behavior in bouillon; nitrate bouillon; Cohn's solution; 
Uschinsky's solution ; milk ; litmus milk ; peptone water in fermen- 
tation tubes with dextrose, with saccharose, with lactose. Can 



THE CUCURBIT WILT: CULTURAL CHARACTERS 139 

you determine the kind of acid produced from cane-sugar? 
Use large flask cultures containing peptone water cane-sugar and 
calcium carbonate. Be certain that they contain only this one 
organism i.e., plate out just before undertaking isolation of the 
acid, and test on young cucumber plants. Study the viscidity 
in cultures : compare it with that of Bacterium leguminosarum. 

Non-nutritional Environment. — Effect of heat, of sunlight, 
of dry air (killed quickly), of chloroform in bouillon, of weak 
acids, of salted bouillons? Can you get any growth in bouillon 
at 9°C., or at 37°C.? How many months will the organism 
live dry on cucumber seeds? 

For the disease. Signs. — Contrast cucumbers and 
squashes. Observe period of incubation; time between local 
appearance of disease on inoculated leaf and general infection 
of the plant. What are the most conspicuous external in- 
dications of this disease? Can you produce it on watermelons? 
on gourds? Write a description of it. 

Histology. — How many centimeters in advance of external 
signs on the inoculated leaf can the bacteria be traced down the 
petiole? Begin before the wilt has attacked the whole leaf- 
blade. With a hot knife sever the petioles where they join 
the stem. Twenty or more plants will be necessary for this 
experiment, which is best performed as soon as a few square 
centimeters of the leaf show the characteristic wilt. After re- 
moval of the inoculated leaf, what per cent of the plants remain 
free from the disease? In tracing the organism from the leaf- 
blade into the stem, determine what tissues are occupied. Are 
cavities formed? Is cellulose destroyed? Is the wilt due to 
lack of water-supply or to toxic action? Are the bacteria in 
the leaf-blade confined to the vessels or do they invade the 
leaf -parenchyma? Do you think they swim through the vessels 
or only grow through them? What are your reasons? 

Make cross- and longitudinal sections of the infected stems. 
If time permits, fix, stain and mount. Which vessels of the 
stem are first occupied? Why? What tissues of the stem other 
than vessels are occupied and destroyed? 

Can you demonstrate the organism in the phloem? In 
the interfascicular parenchyma? Consult Figs. 72 to 78. 



BACTERIAL DISEASES OF PLANTS 




Fig 71 —Surface and buried colonies of Bacillus tracheiphilm (R. 183) ma 
+ 15 agar pouredtplate. Surface wet-shining and smooth. Photographed by 
reflected light. The white spots are high-lights on a perfectly curved surface. 
X 10. 



THE CUCURBIT WILT: HISTOLOGY 141 

Does it ever come to the surface as an ooze on attacked 
plants. Contrast with pear bUght (No. XII, Figs. 282 to 285). 
Make permanent sUdes. Stain with Ziehl's carbol fuchsin. Try 
at least two other stains. 

Variability. — How long does an attacked plant live? 

Have you seen any indications of immunit}^ on the part of 
inoculated plants? of recovery? of slow development of the 
disease? Study this especially in squashes. 






;Mi ^%^ 



t* 





# 



*2/ 



Fig. 72. — Cross-section of a squash petiole from one of my inoculated plants. 
Every bundle is occupied by the bacteria which are stained. They are also to 
some extent in the parenchyma around the bundles. From a planar enlargement 
by the writer. 

What effect, if any, does heavy vs. light watering have 
upon the progress of the disease? Why are not all susceptible 
cucurbits destroyed by this organism? 

Transmission. — If time, season, and location permit, at- 
tempt transmission of the disease by insects. Try Diahrotica 
vitatta or Diahrotica 12-punctata collected from healthy plants. 
Starve 24 to 36 hours, feed on freshly wilting leaves for a short 
time only (early evening), then liberate (over-night, or a less 
time if they have bitten the plants freely) in insect cages con- 



142 



BACTERIAL DISEASES OF PLANTS 




Fig. 73. — Cross-section of a cucuml^er stem (natural infection) showing tlie 
spiral vessels occupied in each bundle by an enormous mass of deei^Iy stained 
Bacillus tracheiphihi>;, 1894. 





-%! 



Fig. 74. — Cross-sect iou of a cucumber stem showing location of Bacillus 
tracheiphilus: 

A. Enlarged view of an inner bundle from Fig. 73, showing the bacterial mass 
confined to the spiral vessels with the exception of two neighboring pitted vessels. 
Around the spirals the bacteria have disintegrated the primary vessel parenchyma, 
forming a small cavity. 

B. An outer bundle of Fig. 73. The occluded spiral vessels are surrounded 
by bacterial cavities; most of the pitted vessels are empty. Photomicrographed 
bv the writer in 1894. 



THE CUCURBIT WILT: TRANSMISSION 



143 




Fig. 75. — Longitudinal section of a cucumber stem, showing complete invasion 
and destruction of the inner part of a bundle by Bacillus tracheiphilus. Photo- 
micrograph by the writer in 1894. 




■^' '- -,.* 













V 



-An 









•-'( 






.••/r*";*^ 
%' 





















Fig. 76. Fig. 77. 

Fig. 76. — Longitudinal section of a cucumber stem exposing two spiral vessels: 
one occupied liy Bacillus tracheiphilus; the other empty. For details of the bacteria 
see a thinner section such as Fig. 77 or 78. 

Fig. 77. — Detail of Bacillus tracheiphilus from a cucumber vessel.- Carbol 
fuchsin stain. 



144 BACTERIAL DISEASES OF PLANTS 

tainiiig thrifty cucumber plants bearing each about 6 or 8 
leaves. Remove next morning. Repot the plants and watch 
closely. The squash bug, Coreus tristis, also may be tried 
(but only on well-grown plants because of the great injury 
done by this bug to small plants). Mr. F. V. Rand believes 
from his experiments that the squash bug does not carry the 
disease. For details on insect transmission see Part I, p. 30). 




Fig. 78. — Cross-section of a cucumber stem such as Fig. 73 showing the in- 
dividual bacteria in a small pitted vessel. This was photographed by the writer 
in 1894 from an unstained glycerin mount using a Welsbach light and an old Zeiss 
non-apochromatic objective the photographic focus of which was not the same as 
the eye focus. The duration of the exposure was about an hour and for this pic- 
ture the negative was enlarged. 

Where does the organism winter-over, i.e., in the soil? 
in hibernating beetles? or on, or in seeds? From its appearance 
on leaves in early summer first in places gnawed by Diabrotica, 
I have long believed that it winters over in the bodies of the 
cucumber beetles. (See recent observations and experiments 
by Frederick V. Rand, of my laboratory, in Journal of Agri- 
cultural Research, Vol. V, November 8, 1915, p. 257; Vol. VI, 
June 12, 1916, p. 417; Dept. Agr. Bulletin No. 828 (Profes- 
sional Paper) 1920 and Phytopathology, Vol. 10, No. 4.) 



THE CUCURBIT WILT! TRANSMISSION 145 

Can you obtain the disease by soil infection (a) through 
wounded roots; (b) through unwounded roots? Contrast with 
No. IV. 

LITERATURE 

For literature, etc., consult: Wilt of Cucurbits in "Bacteria 
in Relation to Plant Diseases," Vol. II, pp. 209-299, Carnegie 
Institution of Washington, 1911, and Ibid., Vol. I (1905) Plates 
3 and 23, and Figs. 8, 9, 13, 14, 21, 47, 48, 68, 69. Also papers 
by Rand, Rand and Enlows and Rand and Cash. 

The first paper on the subject was read by the writer in 
August, 1893, at a meeting of the American Association for 
the advancement of Science (Bot. Gaz., Sept., 1893), This was 
my first contribution to the literature of bacterial diseases of 
plants. The name Bacillus tracheiphilus was first published in 
1895 in Ceniralb.f. Bald. u. Par., II Abt., I Bd., 1895, p. 364. 

II. THE BLACK ROT OF CRUCIFERS 

Type. — This is a common vascular disease of cabbage, cauli- 
flower, kohlrabi, kale, rape, turnips, and mustard. Often whole 
fields are destroyed (Fig. 79). Dr. F. C. von Faber also found 



,-; fS~-J«e4- gi«i»»' ^Tiii<m4 i _ ^) 




Fux. 79. — Cabljage field in Wisconsin, showing all of the plants attacked and 
destroyed by Bacterium campestre. Not a head was harvested. (After Russell.) 



146 



THE BACTERIAL DISEASES OF PLANTS 



it in Germany on winter stock {Matthiola incana). Recently 
Miss Nellie A. Brown, of my laboratory, has found it to be the 
cause of a serious disease of the highly prized Chinese cabbage 
{Brassica petsai L. H. Bailey) grown in the United States. 
There is no reason, however, to suspect that the disease was 
imported from China since it is everywhere in the United 
States. The writer did not succeed in inoculating this disease 
into beans. Infection occurs chiefly through the water-pores 






% 



B 



Fig. 80. 



Fig. 81. 



Fig. 80. — Early stages of water-pore infection on inoculated cabbage leaves: 
A, extrusion of fluid from the water-pores leading to infection; B, appearance of a 
leaf margin about 3 weeks after water-pore infections. 

Fig. 81. — Vertical section through water-pore region of a cabbage leaf show- 
ing bacteria under a stoma. 



on the leaf serratures (Figs. 80 to 84), but may occur also 
through wounds (Fig. 85). Sometimes the leaves wilt, but 
wilting is less conspicuous than in the cucurbit wilt (No. I). 
In the attacked leaf parts there is a conspicuous brown vena- 
tion (Figs. 84, 85) often in a yellow parenchyma. The stem 
bundles are also browned (Figs. 86, 87). It is not a soft rot, the 
attacked leaves becoming dry and leathery rather than wet- 
rotten, unless soft-rot parasites are also present. The black 



THE BLACK ROT OF CRUCIFERS : TYPE 147 

rot occurs almost all over Europe and North America, and 
probably in many other parts of the world. It is said to 
be common in Japan (Gentaro Yamada) and to occur in the 
Philippines (Reinking). It was first reported from Europe 
in 1900 by an American (H. A. Harding). 

Cause. — This disease is due to Bac'erium campestre (Pammel) 
EFS. The organism is a moderately growing, non-viscid, non- 
capsulate, yellow, non-sporiferous, nitrate-reducing, slowly 



k 



V 






V 



I 



Fig. 82. Fig. 83. 

Fig. 82. — Section of a cabbage leaf-tooth parallel to the surface, showing 
two water-pores; one empty, the other blocked by bacteria. 

Fig. 83. — Upper stoma of Fig. 82, enlarged to show more distinctly the rods 
of Bacterium campestre blocking its mouth. 

liquefying, non-gas-forming, casein-precipitating (by a lab fer- 
ment), aerobic, rod-shaped, or slightly curved or club-shaped 
schizomycete, motile by means of a polar flagellum (Fig. 88), 
and forming on the surface of agar-poured plates small circular 
or slightly irregular, wet-shining colonies (Figs. 89 to 91) which 
are pale at first but become distinctly yellow^ (not orange). 
Filamentous chains occur in sugar-rich media. It resists 
drying 12 months or more under favorable conditions (Compare 
with Bacillus iracheiphilus, No. I, and Bacillus carotovorus, 
No. VI, which from bouillon cultures scarcely resist drying 



148 



BACTERIAL DISEASES OF PLANTS 



that number of minutes), and it does not lose its virulence 
readily. Probably it is often 
carried on some of the seeds 
from diseased plants, and 
whole fields may become in- 
fected in this way, directly 
or indirectly. ^ Once present 
in a field, cabbage insects 
greatly favor its distribu- 
tion, especially by gnawing j , 






Fig. 84. Fig. 85. 

Fig. 84. — -Water-pore infections on a cabbage leaf, 2 months along. Pure- 
culture inoculation by the writer. Bacterial movement downward in the vessels. 

Fig. 85. — Central part of a cabbage leaf showing brown venation due to 
Bacterium campestre. The inoculation was on a leaf lower down. Bacterial move- 
ment downward in vessels of the inoculated leaf, then upward, first in vessels of 
the stem and afterward in those of this leaf. District of Columlaia, 1897. 



into diseased leaves and then crawling over healthy ones. 

1 Geo. K. K. Link has reported to me verbally a case observed by him in Southern 
Florida on new land in the spring of 1919 where 60 per cent of 100 acres of cabbages 
was destroyed by this disease, the total shipments amounting to only 40 car-loads. 



THE BLACK ROT OF CRUCIFERS : TECHNIC 149 

Technic. — The organism is easily isolated on agar-poured 
plates inoculated from suitable material, e.g., cabbage petioles. 
There are no special difficulties except that sometimes heavy 
sowings are necessary when the organism has been in the tissues 
a long time. If difficulties are encountered, the second method 
described under No. I may be tried. The bacteria grow readily 
on all ordinary culture-media; steamed potato is a good sub- 
stratum. 



^. 



^^ 



B 




- 




■ /t 


i y ^ 


* 


, •'. 


\^ 






1 


t 


V 


■a 




■^ 


X 




■v 


,»4f 


'^, 


'-• t. 


./, ■ 


ft' 

V 


• ^ 






Fig. 86. Fig. 87. 

Fig. 86. — A. Cross-section of a cabbage petiole, showing only two infected 
and blackened bundles, and correspondingly only a small area in the center of the 
blade of the leaf was diseased. 

B. Cross-section of a cabbage petiole showing every bundle blackened by 
Bacterium campestre, and correspondingly all of the leaf-blade was diseased. Same 
series of inoculations as A, 1915. 

Fig. 87. — Section of the fleshy part of a kohlrabi showing black bundles due 
to Bacterium campestre. Collected by the writer in Florida in 1904. 

For inoculation purposes use young plants of cabbage, turnip 
or cauliflower. The same hothouse cultural directions apply as 
for cucumbers, etc., under No. I. 

For contrast the resistant Houser cabbage may be used. 
Several hundred plants should be provided, and they must be 
kept free from insects and molluscs. The seed should be sown 
at least six w^eeks before the plants are needed. 



150 



BACTERIAL DISEASES OF PLANTS 



Inoculate by needle-pricks on the under surface of the fleshy 
petiole, some petioles on one side only, some on both sides and 

k A B / 





% 



Fig. 88. — A, B. f lagella of BacUriu/n catnpestre. Stained by van Eiineiif^em's 
silver nitrate method. X 1000. 




Fig. 89. — A, Surface colonies of -Badermm ram/jesire on nutrient agar. Plated 
fromJlD.C. rape. Time, 6 days. Photo Feb. 6, 1919. X 10. B, The same by 
oblique transmitted light. 

in the middle, setting the needle deep enough to enter the bun- 
dles. Other plants should be inoculated in the upper soft stem 



THE BLACK ROT OF CRUCIFERS: TECHNIC 



151 




# a 9 




, X 





Fig. 90.— Surface and buried colonies of Bacterium campestre {^ew York iso- 
lation of 1908) at end of 10 days in +14 beef peptone agar. Crystal at x. Colo- 
nies coming to the surface at a, b. Photographed February 11, 1919, by direct 
transmitted light. X 10. Tested on Crucifers Feb. 9, 1919. 



152 



BACTERIAL DISEASES OF PLANTS 




|PiQ 91.— .4, B. Surface and buried colonies of Bacterium campestre (isolated 
from D. C. rape) at end of 13 days on + 14 beef-peptone agar plates. Four crystals 
are present, 3 in colonies. X is a buried colony coming to the surface. Photo- 
graphed by transmitted oblique light February 13, 1919. X 10. 



THE BLACK ROT OF CRUCIFERS : TECHNIC 



153 



immediately under the origin of a leaf; still others in the paren- 
chyma of the leaf-blade — some at the apex, others on one side 
midway down. Select leaves several removes from the lowest, 




Fi(i. 92. — Cultures of Bncterium cnmpestre (B), and Bacterium phaseoli (A) in 
Dunham's solution at end of 4 days. 

otherwise the leaf may be unjointed and thrown off before 
stem-infection has occurred. 

The plants should be well watered and growing freely for 
best results, which should begin to be visible in 10 days, more or 
less, from the time the punctures are made. 




Fig. 93. — Sheaf-like tyrosin crystals in a 2-months 12-(lay old litmus-milk culture 
of Bacterium cnnipestre. X 6. 

For water-pore infections confine young plants in an infec- 
tion cage in a cool place and spray freely wdth a water suspension 
from a 48-hour agar or potato-streak culture. Keep the plants 



154 



BACTERIAL DISEASES OF PLANTS 



in the cages in moist air for 30 hours and water freely. Examine 
the plants morning, noon and evening, and if the leaves look dry 
atomize sterile water on them and flood the soil around the 




Fig. 94. — Cross-section of a cauliflower stem sliowing a cavity in a small bundle 
occupied by Bacterium campestre. 



Vi 



- II il« j^Vk I. 'Vj '— **■' 
\Y •l> »" A* ' \ 



cages. Examine the leaf serra- 
tures at least once every 3 days 
until signs appear. 

Try soil infections through 
broken roots: a, on very young 
plants; b, on plants having stems 
1 4 to 1 2 inch in diameter. What 
do you conclude? 

Determine 



For the organism. Mor- 
phology. — Size in microns, form 
(straight rods, curved rods, club- 
shaped rods), aggregation of 

1'|fl^'"'V^ v3*^^ ^^^"^®^^^' ^■^■' ch^i^^' filaments, 

iM^Mi^^M^^S^^I^ etc., motility on margin of 

hanging drop, absence of endo- 
spores, presence and distribu- 
tion of flagella (Lofiler's stain). 
Is there ever more than one 
fiagellum? Try Gram's stain. Look for involution forms. 
Use various media. Try cultures of different ages. 



Fig. 95. — Longitudinal section of 
a cauliflower bundle, similar to Fig. 
94, showing entire disorganization due 
to Bacterium catnpesire. 



THE BLACK ROT OF CRUCIFERS : CULTURAL CHARACTERS 155 

Cultural Characters. — ^Thin-sown agar plates (Figs. 89 to 91), 
streaks and stabs; gelatin, ditto. Behavior on steamed potato- 
cylinders standing in water (contrast with No. III). Why is 
the growth on potato so prolonged? Test for reducing sugars. 
Growth in Dunham's solution contrasting with Bacterium phas- 
eoli (Fig. 92). Behavior in bouillon, nitrate bouillon, Cohn's 
solution, Uschinsky's solution, milk, lavender-colored litmus 
milk (Fig. 93), peptone water in fermentation tubes with all 
the common sugars and alcohols. Can you find an^^ that will 







Fig. 96. — Cross-section of the root of an inoculated turnip showing Bacterium 
campestre occupying a small bundle. Cell walls swollen. X about 600. 

induce it to grow in the closed end of a properly constructed 
fermentation tube? What is the nature of the yellow pigment? 
Try extraction with solvents — alcohols, ethers, chloroform, ben- 
zine, benzole, carbon bisulphide, etc. 

Non-nutritional Environment. — Can you obtain growth in 
bouillon at 9°C. and at 37°C.? Determine maximum tempera- 
ture for growth, and minimum; resistance to dry air (on cover 
slips, or silk threads, and on baked turnip or cabbage seed); 
effect of chloroform in bouillon (contrast with No. Ill); killing 



156 



BACTERIAL DISEASES OF PLANTS 



power of sunlight (in thin-sown agar plates); effect of weak 
acids and alkalis; of salted bouillons (contrast with Nos. I and 
III). Vitality on culture-media. 

For the disease. Signs. — Determine time between inocu- 
lation {a, by needle pricks — b, through the water-pores) and 
first local signs of the disease. In case of plants inoculated on 
the leaf -blade, how long before signs appear in other leaves? 




Fig. 97. — Like Fig. 96, but. shows l)egiiimng oi a cavity. X 500. 

Contrast inoculations on plants growing rapidly and slowly. 
In case of inoculations on the petiole, how long before signs 
appear in its blade? After the infection (blackening) of the 
water-pore region, how long before the veins of the leaf begin 
to show the dark stain? Describe the disease. 

Histology. — Determine by sections the occupation of the 
substomatic region in the infected leaf-teeth. Can you trace 



THE BLACK ROT OF CRUCIFERS: HISTOLOGY 157 

the organisms from the water-pore region into the veins of the 
leaf? Try microtome sections. Make preparations showing 
the bacteria in the vessels of the leaf-blade. How soon after 
water-pore infection can they be detected in the veins of the leaf? 
Have you observed them forming cavities in the leaf-paren- 
chyma around the bundles? Why do they not produce a soft 
rot of the leaf? Does the organism ever enter the leaf through 
ordinary stomata? Why not? How many centimeters in 
advance of the brown stain can you trace the bacterial invasion 
downward in the leaf ? Have you seen any indication that special 
areas of leaf venation anastomose with special leaf-traces of the 
petiole? 

Observe in stems of cabbage an increase of chlorophyll 
around the diseased bundles. What causes it? (Compare with 
leaf spots of No. VIII and with tumors of No. XIV on Paris 
daisy) . 

Where is the brown stain located? Can you reproduce it 
in culture-media? What is its nature? Is it a humus com- 
pound? 

Stain sections of infected leaf and stem, using nigrosin, 
basic fuchsin, or iron hematoxylin. Make permanent prepara- 
tions. 

Do the infected vessels contain masses of granules independ- 
ent of the bacteria? What are these? Are they Lohnis' 
granules? Cut the vessels longitudinally. 

What is the action of the organism on the tissues? Is 
there a toxic action distinct from a solvent one? Is the cell- 
wall destroyed? What then becomes of it? Consult Figs. 94 
to 99. 

Can you find the organism in the roots of cabbage or cauli- 
flower? Is it commonly a root-infection? Does the organism 
commonly ooze to the surface of attacked plants? (Compare 
with Nos. VIII, X, XI, XII and XIII.) 

Variability. — How long does the attacked plant live? Com- 
pare early and late infections; inoculations on slow-growing 
pot-bound dwarfed plants with those on rapid-growing plants. 
Study the functioning of water-pores as related to infection. 



158 



BACTERIAL DISEASES OF PLANTS 




Fig. 98. — Cross-section of a turnip root showing a parenchyma cell with Bacterium 
campestre filling the intercellular spaces. X 1000 circa. 




!'''^ 




Fig. 99. — Longitudinal section of root of an inoculated turnip showing Bac- 
terium campestre occupying a vessel and cells at the left. X circa 800. Thejin- 
oculation was made on the leaves by needle pricks. 



THE BLACK ROT OF CRUCIFERS: TRANSMISSION 159 

Transmission. — Greenhouse slugs may be used, feeding them 
first on infected leaves and then on sound plants. Also larvae 
of the cabbage butterfly. Can the disease be spread by aphides? 
If opportunity exists, collect seeds from diseased plants and 
try to isolate the organism from them (rather difficult) and to 
get infected seedlings from them. Why should a seedsman 
collect and disseminate seeds from stock he knows to be diseased? 

The writer has seen a serious outbreak of the disease on 
parts of a cabbage field that received as a manure the diseased 
refuse from a storehouse in which brown-rotted cabbage had been 
wintered over (see No. VII). He has seen an entire crop ruined 
and the organism introduced into the soil of a field previously 
free from it by setting it out with plants from an infected seed 
bed. (See U. S. Dept. of Agr., Farmers' Bull. No. 68.) 

MEANS OF PREVENTION 

Use of seed derived from healthy plants. Seed beds on 
land free from the disease. Care in transplanting that roots 
shall be wounded as little as possible. 

LITERATURE 

For literature, etc., consult: Black rot of Cruciferous Plants 
in "Bacteria in Relation to Plant Diseases," Vol II, pp. 300-334, 
Carnegie Institution of Washington, 1911. See also Ibid., 
Vol. I, Figs. 4, 5, 6, 7, 18, 19, 76, 77, 78, 79, 87, 115, 116, 117. 

The first important paper on the subject was published 
in 1893 by Prof. L. H. Pammel (Bot. Gaz., Jan., 1893). The 
organism was first named Bacillus campeslris by Pammel in 
1895 in Bull. No. 27, Iowa Agr. Col. Exp. Station, pp. 130-134. 

The last paper is by Walker and Tisdale: Observations on 
Seed Transmission of the Cabbage Black Rot Organism. Phyto- 
pathology, Vol. 10, No. 3, March, 1920, pp. 175-177. 

These authors have proved introduction of the disease into \\ isconsin on seed 
imported from north Europe. They have also estabhshed that the disease can 
be reduced to negHgible proportions by soakiufj; the seed for 'SO minutes in 1-1000 
mercuric chlorid water. 



III. STEWART'S DISEASE OF MAIZE 

Type. — This is a vascular disease confined principally to 
sweet corns, especially those rich in sugar and ripening early, 
but it has been seen by the writer upon several varieties of 
field corn. The foliage shrivels gradually, the lower leaves 
usually; first (Fig. 100); the male inflorescence develops pre- 




FiG. 100. — Large sweet-corn plant destroyed by Aplanobader stewarti. A 
natural infection. Bundles of the stem occupied by the yellow slime. District 
of. Columbia, 1903. 

maturely and is white (Fig. 101); and on cross-section or lon- 
gitudinal-section of the stem a yellow slime oozes from the 
vascular bundles (Figs. 102 and 103); stooling also sometimes 
occurs (Fig. 104). Infection takes place principally in the seed- 
ling stage through stomata and is greatly favored by actively 
functioning water-pores situated on the young leaf-tips. The 

IGO 



Stewart's disease of maize: type 161 

organism is extremely abundant in the vessels and is much 
inclined to come to the surface of the husks through stomata 
(Figs. 105, 106, and 107) ; thus flooding the kernels, but it may 
occur also inside the kernels, particularly at their junction with 
the cob (Figs. 108 to 111). Some of the infected plants are de- 
stroyed in the seedling stage (Fig. 112), but many of them reach 
a height of several feet before showing secondary signs (Fig. 
113). It is a typical example of a seed-borne infection. Noth- 
ing is known as to the occurrence of this disease outside of the 
United States. Miss Doidge has not seen it in South Africa. 
The exact distribution of the disease in the United States is un- 
known but it occurs from New York and Maryland to California. 

Cause. — It is due to Aplanobacter stewarti (EFS) McC. 
This is a non-motile, non-flagellate, non-sporiferous, inadhesive, 
or moderately viscid, yellow, slow-growing, non-liquefying, 
non-milk-curdling, non-nitrate-reducing, non-gas forming, non- 
starch-consuming, chloroform-tolerant, sodium chlorid-tolerant, 
aerobic, rod-shaped schizomycete, growing on the surface of 
agar-poured plates in the form of small, flat, circular or nearly 
circular pale colonies which become yellow with age.^ It 
reddens lavender-colored litmus milk slightly and does not 
grow in Cohn's solution. Its growth on steamed potato is 
thin and soon at an end (contrast with Nos. II, VIII, or X). 
Why is this? In Dunham's solution containing methylene 
blue the bacterial precipitate should be blue. 

Its minimum temperature in +15 peptone beef bouillon 
is above 9°C. At this temperature there was no clouding 
in 14 days. The checks at room temperature clouded heavily 
the second day and formed a pellicle the third day. It is not 
sensitive to dry air, and like No. II retains its vitality and its 
virulence for a long time. It is rather tolerant of weak organic 
acids. On the kernels the majority of the bacteria are destroyed 
by exposure for 15 minutes to 1 :1000 mercuric chlorid water: 

1 Sometimes the surface colonies on agar have depressed centers (Fig. 1145). 
No mention was made of these in Volume III of my monograph because I was not 
then certain that they belonged in the life-cycle of this organism, but recently 
Lucia McCuUoch, of my laboratory, has proved them to be infectious. She has 
also proved my former statements respecting the motility of this organism to be 
incorrect (see Phytopathology, August, 1918, p. 440). 



IQ2 BACTERIAL DISEASES OF PLANTS 




M 



'/' 



Fig 101 -Blue Squaw fln.t corn from a field on Arlington Farm, July 16. 
1915 (an early sort) : No. 1, slightly diseased ; No. 2, badly diseased, showmg white 
to?(prLatte^ dead male mflorescenee) and dry pale leaves, due to Aplano- 
bacier stewarti. Photo by James F. Brewer. 



STEWARTS DISEASE OF MAIZE: CAUSE 



163 



first plunge the seeds momentarily into alcohol, rinse them 
very lightly and dry quickly or plant at once. Why this last 
direction? Why also first into alcohol? It fills the infected 
vascular bundles with a yellow slime which oozes on cross- 
section (unless the plants have been frosted). Why not then? 






Fig. 102. — Cross-section of a diseased sweet-corn stalk showing Aplanobncier 
stewarti oozing from the bundles. Planar enlargement. 

Technic. — Isolations may be made from externally sound 
upper internodes of the maize stem, by the first method de- 
scribed under No. I. Often pure cultures may be obtained 
directly from the cut stem by streaks on steamed potato or 






Fig. 103. — Water mount of a sweet-corn stem m longitudinal section showing 
Aplanohacter stewarti oozing from a vascular bundle like smoke from a chimney. 
(After F. C. Stewart.) 

nutrient agar if the surface sterilization has been thorough, 
but if so made, subsequently they should be plated out. It 
is more difficult to isolate from the interior of kernels. Such 
kernels should be soaked in 1:1000 mercuric chlorid water 
for 30 to 60 seconds, to inhibit, rather than to kill, surface 



164 



BACTERIAL DISEASES OF PLANTS 



organisms. The bases may now be removed, crushed in a sterile 
mortar and allowed to soak in bouillon for some hours before 
plates are poured. Some of the latter should be sown heavily. 
Keep the tubes and pour a second set of plates next day; pour 





Fig. 104. Fig. 105. 

Fig. 104. — Corn plant, showing very pronounced dwarfing, premature develop- 
ment of male inflorescence and stooling due to Aplanobacter dewarti. Vessels 
full of the yellow slime. From Chula Vista, California, in 1915. 

Fig. 105. — Spots on inner husk of a sweet-corn ear as a result of bacterial cavi- 
ties due to Aplanobacter stewarti. Plant from infected seed. Spots bright 
yellow. 

also from dilutions of the same, if clouding has developed. There 
are various non-parasitic, motile, yellow schizomycetes on the 
surface of corn kernels, and only those non-motile forms which 
behave properly in the agar, gelatin, nitrate bouillon, litmus 



Stewart's disease of maize: technic 165 

milk, Uschinsky's solution and Cohn's solution need be tried 
further. 

For inoculation purposes select first of all seedling plants 
and inoculate from young potato or agar streaks on the leaf- 
tips when the plants are 2 to 3 inches high and show only 
2 or 3 unfolding leaves. The inoculations may be made by 
spraying or by touching the leaf-tip with an infected platinum 
needle. After inoculation the young plants may be placed 






•'"M- 










Fig. 106 — Corn husk in cross-section sliowing vessels and intercellular spaces 
of the parenchyma (dark areas) filled with Aplanobacter stewarti. Stoma oozing 
bacteria at A". See Fig. 107. 

either in cages or under the greenhouse bench. The essential 
is damp earth and a moist shaded place where the water-pores 
at the leaf-tips will function freely. Examine from time to time 
to make sure that drops of water remain on the leaf-tips. If nec- 
essary, wet down the greenhouse thoroughly so as to saturate the 
air. After 30 hours set on the bench and withhold water for a 
day, if the soil looks wet. Change the plants frequently to larger 
pots and transplant into the garden at the end of June (May in 
the South) when the plants are about 15 inches tall, and make 



166 



BACTERIAL DISEASES OF PLANTS 




¥t.'. 






.^- 



FiG 107 —A detail from Fig. 106 X showing Aplannhackr deiva rti sepavatmg 
cells of the corn husk and filUng the substomatic chamber. Photomicrograph by 
the writer. 



Stewart's disease of maize: technic 167 

the final examinations in September before frost supervenes. 
One or more of the following sensitive varieties may be used: 
Black ^Mexican, Colden Bantam, Crosby's Early, Cosmopolitan, 
Pocahontas. Along with these, white and yellow field corns 
should be inoculated for comparison, taking pains to secure 
the names of the latter. The seedlings should be ready for 
inoculation about 8 days after planting, which, in the North, 
should be toward the end of May, if the seedlings are to be 
transplanted into the open field. 

Uninoculated check-plants should be held. These should be 
grown at some distance from the inoculated plants (preferably 




W 



Fig. 108. — Cro.s8-secti«)n (jf a small hiuullc at the extrriiic base of a kernel of 
sweet colli showing: AplaKolxirwr .stcwarti in the single vessel. 

in an adjoining house) and should be transplanted to the other 
side of the garden. Even then, some cases may be expected 
unless the seed corn is beyond suspicion, and the house free from 
insects. 

Determine 

For the organism. Morphology. — Size in microns, form 
(Ziehl's carbol fuchsin or amjd Gram may be used for staining), 
aggregation of elements, motility (hanging drop), question as 
to occurrence of flagella (hanging drop and van Ermengem's 
silver-nitrate stain; in case of the hanging-drop method, boil the 



168 



BACTERIAL DISEASES OF PLANTS 



culture and reexamine), absence of endospores (heat, stains), 
presence or absence of capsule, occurrence of involution forms, 
reaction to Gram's stain (diaphragm wide open). 

Cultural Characters. — On agar (Figs. 114, 115), on gelatin, on 
steamed potato, in bouillon, nitrate bouillon, Cohn's solution, 
Uschinsky's solution, lavender-blue litmus milk. Fermenta- 
tion tubes in peptone water with various pure sugars and alcohols. 




Fig. 109. — A larger bundle at the same level as Fig. 108, showing Aplanobacter 
steivarti occupying many of the vessels. 

Growth in acid plant juices, e.g., green tomato juice full strength 
and diluted with an equal volume of water (titrate with phenol- 
phthalein and N/20 sodium hydrate to determine the acidity). 
Compare with No. II or No. VIII. 

Non-nutritional Environment. — What is the optimum tem- 
perature for growth? the maximum? the minimum? Can 
you get any clouding of +15 peptone beef bouillon at 9°C.? 



Stewart's disease: non-nutritional environment 169 

Contrast with No. IX. Effect of sunlight? of dry air? of 
freezing? of weak acids? of weak sodium hydrate? of chloro- 



m 





t^ 




Fig. 110 — Longitudinal section of outer layers at base of a sweet-corn kernel 
(level of the radicle) showing presence of A planobacter stewarti hciween cell-walls 
and in the vessels. 




Fig. 111. — Aplanobact( )■ 
corn kernel. 



^/( iriuii loiniing a cavity in the periphery of a sweet- 
Same section and same level as Fig. 110. 



form in bouillon? Maximum toleration of sodium chlorid in 
bouillon? (Begin with 5 per cent. Contrast with No. I.) 

For the disease. Signs. — How soon does the disease ap- 
pear in the inoculated leaves? How many days between the 



170 



BACTERIAL DISEASES OF PLANTS 



local appearance of the disease on the leaf -tips and signs of gen- 
eral infection in the plant? On well-grown plants the earliest 
signs are flagging and shriveling of the lower leaves, and "white 
top," i.e., the premature development and drying-out of the 
male inflorescence. This is conspicuous at a distance (Fig. 101). 
Watch for these signs and try to correlate them with the pres- 
ence of bacteria in the vascular system of stem and leaf. 

Can you find any macroscopic 
evidence of the presence of the 
disease on the inside (surface) of 
the leaf-sheaths? or in the ear, 
especially on the husks? Contrast 
with Nos. I and II. 

Look for dwarfing effects. Is 
the plant as tall as its fellows? 
Are the ears well filled? Are the 
roots generally sound? Do the 
leaves become yellow before they 
dry out? Does the plant rot? or 
break over? Describe the disease. 
Histology. — Select, section and 
stain (in Ziehl's carbol fuchsin) a 
number of leaf-tips, some days 
after inoculation (4 to 7 days). 
Can you find distinct evidences of 
infection? Have the bacteria en- 
tered through ordinary stomata, 
or through the water-pores? 

Cut and stain cross- and longi- 
tudinal sections of infected stems. 
What vascular tissues are most 
subject to attack? Does the organism attack the phloem? 
Do cavities occur uniformly? In maturing plants how far can 
you trace the vascular infection? In the vessels of the stem is 
the general movement of the bacteria upward or downward? 
What is your reason for this belief? In large leaves begin- 
ning to wilt or shrivel is there simply occlusion of the stem- 
bundles which supply these leaves, or are the bacteria also 




Fig. 112. — Young sweet-corn 
plant inoculated on tips of the 
leaves in the seedling stage and 
promptly destroyed by Aplatto- 
bacter stewarti. District of Colum- 
bia, 1902. Time, 19 davs. 



STEWARTS DISEASE OF MAIZE: HISTOLOGY 



171 



present in the leaves themselves? Are such large leaves ever 
infected first at the apex? Make cross-sections: a at the tip; 
b near their junction with the 
stem; and c in the middle of the 
leaf. Try several leaves. 
Have you seen any evidence of 
wound-infection? If diseased 
ears are available, study care- 
fully by means of sections at 
different levels the upward and 
outward movement of the bac- 
teria in the pedicle, cob and 
husks. Try to demonstrate vas- 
cular infection in the base of 
the kernel. 

Observe yellow pockets in 
the pith and in the husks. 
Make sections and determine 
the contents of these yellow 
spots. Draw what you have 
seen. 

Have you observed any 
brown stain in the stems? Is it 
local or general? Where does 
it first begin? Is this stain a 
host reaction ? Does the organ- 
ism cause a brown stain in any 
culture medium? Compare 
with Xo. II and No. X. 

What impresses 3'ou most 
about this disease? Do you 
think there are any toxins liber- 
ated? What are your reasons? 
How many bacteria would you 
estimate to be present in an in- 
fected well-grown plant when 
the leaves are beginning to shrivel? What proportion of the 
vascular bundles are occupied? Is there anything correspond- 




FiG. 113. — Sweet-corn plant de- 
stro3'ed by Aplanobacter stewarti as a 
result of an inoculation on the leaf- 
tips in the seedling stage. Plant 
dwarfed and vessels full of the yellow- 
slime. Time, more than 60 dajs. 
District of Columbia. 1902. 



172 



BACTERIAL DISEASES OF PLANTS 



ing to this vascular invasion in the animal world? Consult Figs. 
116 to 118. 

Variability. — How long does an attacked plant live? In 
what varieties of field corn have you found the disease, or been 
able to cause it by inoculations? Try many varieties, if you 




Fig. 114. — A, Agar poured plate surface colonies of Aplanobacter stewarti, B, Type 
showing pitted centers. X 10. 

have opportunity, and make records. There is much to be 
learned about the occurrence of this disease in field corns. 
What occurs when, by needle-pricks, you inoculate sweet-corn 
plants 2 or 3 feet high on the upper leaf-blades rather than on 
the leaf-tips in the seedling stage? What does this teach you? 
Can you check the progress of the disease by allowing in- 



Stewart's disease of maize: variability 173 



% 








H 





%■ 



« 



IP 



/r 



/ 



^.»<aiis£iiatiMi ^ 



_ Fig. 115.— Typical buried eoluiues of Aplanubacltr .steLcurti (Kaud\> No. 4U8) 
in + 14 beef peptone agar plate, one coming to the surface. At ;r, x, x, crystals 
Plate 8 days old at about 23°C. X 10. 



174 



BACTERIAL DISEASES OF PLANTS 



oculated plants to become potbound? Can you cause general 
infection by stem inoculations of half-grown plants? Inocu- 
late in the middle of internodes and also close under nodes. 
Does nodal inoculation make any difference, i.e., hasten 
infection? 

If you have opportunity, examine infected fields and make 
lists of susceptible and resistant sweet corns. Make extensive 




#% 
CP 



:'^ 



Fig. 116. Fig. 117. 

Fig. 116.- — Longitudinal section of two sweet-corn stems showing a brown node 
and yellow stripes in the internodes, and a bacterial cavity in the middle of the 
left-hand stem. This cavity was bright yellow. Sections from plants inoculated 
in the seedling stage. 

Fig. 117. — Stained section of such astern as Fig. 102 enlarged showing two 
bundles occupied by Aplanobacter stewnrti and two free. 



counts and express your data in per cents. Reexamine your 
fields some weeks later for additional cases. 

Remember: none of the diseases described in this book are 
known thoroughly and you may have opportunity to add to the 
sum of our knowledge. 

Transmission. — This is commonly by way of seed corn. 
Very likely also by way of soil previously infected. Read what 



Stewart's disease of maize: transmission 175 

is said upon this subject in '^Bacteria in Relation to Plant 
Diseases," Vol. Ill, pp. 114-127, and if you can find gardens or 
fields in which this disease has appeared for the first time, pro- 
cure seeds of the infected varieties from the same seedsman and 
of the same origin, and repeat the experiment, trying also (as 
mentioned under Technic) to find the organism in some of the 




Fig. lis — Single infected and disorganized l)undle of sweet-corn stem much 
enlarged, showing the bacterial mass of ApUmobacter steirnrti dark because it was 
stained red with carbol fuchsin. 

kernels (the vessels toward the base) by means of stained sec- 
tions and by the agar-poured-plate method. Tell the seedsman 
the result. 

The disease occurs in the United States every year in many 
localities where sweet corns are grown for market and the organ- 
ism may therefore be supposed to winter over in the soil, but 
in places where it appears for the first time it may be assumed 



176 BACTERIAL DISEASES OF PLANTS 

to have been brought on infected seed. No one has yet isolated 
it from the soil, nor do we knoio that it persists in soils. 

MEANS OF PREVENTION 

This disease is disseminated almost exclusively by the seed- 
trade. When growers of seed-corn have learned to recognize it 
and refuse to market corn from fields in which it has been 
prevalent, the disease will almost entirely disappear. Mean- 
while, seed-corn may be rendered nearly free from the organism 
by plunging it for a moment into alcohol and then for 15 
minutes into 1:1000 mercuric chlorid water, whereupon it may 
be rinsed in water (for a moment only) and then spread out to 
dry, or planted at once. 

LITERATURE 

Read Stewart's BuUetin No. 130, Geneva, N. Y. Experi- 
ment Station, which is the first paper. 

Consult Stewart's Disease of Sweet corn (Maize) in ''Bac- 
teria in Relation to Plant Diseases," Vol. Ill, pp. 89-147; also 
Ibid., Vol. I, Figs. 1, 73, 74, 75; and Vol. II, Fig. 14 and Plate 
17. The organism was first named by the writer Pseudomonas 
stewarti in 1898 in. Proc. Am. Asso. Adv. Sci., Vol. XLVII, 
pp. 422-426. 



\ 



I 



IV. THE BROWN ROT OF SOLANACEAE 

Type. — This is a destructive parenchymo-vascular wilt dis- 
ease of wide distribution on a great variety of plants (Figs. 119 
to 128. 

It was first described by the writer from the potato, tomato 
and eggplant in 1896 and subsequently from tobacco (1908), but 




Fig. 119. — Potato attacked by Bacterium solanacearum. From a field near Wash- 
ington, D. C. 

it occurs also in Physalis and red peppers, and outside of the 
Solanaceae in species of Urticaceae, Leguminosae, Verbenaceae 
and Compositae (Honing). Fulton and Winston found it on 
peanuts in North Carolina (1913). It has been inoculated suc- 
cessfully into Sesamum (Honing), and it also attacks the nas- 
turtium (Mary Katherine Bryan, Jour. Agric. Res., Vol. lA , pp. 

12 177 



178 



BACTERIAL DISEASES OF PLANTS 



451-458, 1915). Stanford and Wolf have found it attacking 
several weeds in North Carolina (Eclipta alba, Ambrosia artemis- 
iaefolia) and have successfully inoculated it into a variety of 
plants including Impatiens balsamina. In 1918 fields of Rici- 
nus coinmunis (castor oil plant) were attacked by it and seriously 
injured in Georgia and Florida (Smith and Godfrey, I. c.) In our 




Fig. 120. — Early Rose potato inoculated by needle-pricks on the stem with a 
culture of Bacterium solanacearum. Time, 37 days. Wilt developed slowly, 1905. 

hothouse tests we found it able to attack Vanilla planifolia, Heli- 
anthus annuus and young cotton plants. In 1919 it was found 
in Florida on beans (Smith and McCuUoch, I. c.) and was suc- 
cessfully inoculated into beans and peas. 

On tomatoes the leaves are bent downward (Fig. 121, left) 
and the stems develop numerous incipient roots (Fig. 122, left). 
To a lesser extent these roots appear on tobacco|stems. They 



THE BROWN ROT OF SOLANACEAE : TYPE 



179 



occur also on nasturtium stems. On tobacco, longitudinal black 
sunken stripes appear on the softer stems and large, irregular 
brown spots on the leaves, especially on the basal ''ears" of 
the leaf. The tobacco leaves also frequently show brown veins. 
Compare with the Black Rot of Crucifers (No. II). 




kssy 



Fig. 121. Fig. 122. 

Fig. 121. — Young tomato plant: left-hand shoot inoculated at A' by needle- 
pricks introducinp; Bacterium solanacearum; right shoot pricked with a sterile 
needle. Photographed at end of 5 days. Terminal leaves of inoculated shoot 
wilting and lower leaves reflexed. The inoculated stem begins to show incipient 
roots while the other is free (see Fig. 122). 

Fig. 122.— Part of left and right branch of Fig. 121 after 12 days. The 
branch showing the incipient roots was inoculated on the two nodes immediately 
above the part here shown. The smooth branch was pricked with a sterile needle 
in the internodes here shown. 

On all of the host plants, the foliage wilts more or less, often 
completely, the occluded vessels are usually stained brown or 
black, and there is often an extensive destruction of pith and 
bark, so that in tomato and tobacco the stem may be honey- 
combed for long distances with bacterial cavities. Sometimes 



180 



BACTERIAL DISEASES OF PLANTS 



the alkaline slime oozes to the surface but often the surface is 
sound. On potato, narrow dark stripes corresponding to the 
infected bundles often show through leaves and stems. On the 
leaves of the potato, infection may be lateral, running out on one 
side of the petiole and in particular veins of the leaflets as a 
black stain: compare with Fire Blight of the Pear (No. XII). 
The tubers are also subject to infection, principally in the vascu- 




FiG. 123. — ^Tobacco leaf wilted by Bacterium solanacearum. 
inoculation made bv the writer in 1906. 



Results of a stem 



lar region and usually by way of the stem, through the vessels 
of the rhizome; cavities are formed and these rupture to the 
surface. On cross-section of tubers in early stages there is, 
especially at the stem end, a ring of brown stain and a gray bac- 
terial ooze limited to the outer (vascular) part of the tuber 
(Fig. 129). This stain often gives to portions of the surface of 
the tuber a dusky hue even when the skin is unbroken and the 
outer tissues are sound. Stems of attacked Helianthus, Tro- 



THE BROWN ROT OF SOLANACEAE : TYPE 



181 



paeolum and Impatiens also have a livid color (see Fig. 1265) 
and ooze bacteria through rifts and stomata or on cross-section 
(Figs. 130 and 212B). 

It is commonly a wound-infection disease, but infection may 
also occur through stomata. More often than not the bacteria 
enter the plant underground, through broken or punctured roots. 
It is easy to obtain the disease experimentally on Datura, Tro- 




FiG. 124. — Young tobacco plant inoculated with Bucttrluui sulunaccarutn bj^ 
needle-pricks and photographed at the end of 17 days. The bundles of stem and 
leaves were browned and swarming with the bacteria. The plant showed marked 
dwarfing before it wilted. Inoculated May 1, 1915, with " Creedmore " of lessened 
virulence. Plated in 1914 from North Carolina tobacco. Photographed May 17. 
Check plant at left. 

paeolum, and Ricinus by means of broken roots. The disease 
is very apt to occur in carelessly transplanted seedlings or in 
plants whose root-system is attacked by parasitic nematodes 
{Heterodera radicicola) , or is gnawed by insects. 

It is a disease in which there is often a conspicuous dwarfing 
of attacked parts (Figs. 121 X, 127 and 131). Young soft tissues 
are more subject to the disease than those containing less water. 



182 



BACTERIAL DISEASES OF PLANTS 




Fig. 125. — Dwarf nastur- 
tium plant wilted by Bacter- 
ium solanacearum. From a 
garden in Baltimore in 1914. 
(See paper by M. Katherine 
Bryan in Journal of Agricul- 
tural Research, August, 1919, 
p. 451.) 



It prevails under the equator and 
in the North and South Temperate 
Zones, but its northern and southern 
distribution are unknown. It seems to 
thrive best in the Eastern United States 
on washed river sands. In the Old 
World it is believed to occur from Japan 
and the Philippines to New Zealand 
and westward through Java, Sumatra 
and India to South Africa and Italy (?). 
In the United States it occurs from 
Maryland and New Jersey south to 
Florida, Cuba and Porto Rico, but its 
northward and westward distribution 
in this country are unknown. It has 
not been reported from South America 
but undoubtedly it occurs there. Prob- 
ably its range is that of the plants sub- 
ject to it. 

Cause. — This disease is due to Bac- 
teriuin solanacearum EFS. This is a 
non-viscid, motile, polar flagellate, white 
(bluish, brownish, opalescent), non- 
sporiferous, slow-growing, non-lique- 
fying (or very slowly liquefying?), non- 
starch-destroying, aerobic, milk-clearing 
(non-curdling) nitrate-reducing, non- 
gas-forming, rod-shaped schizomycete, 
forming on the surface of agar-poured 
plates small circular, or rather nearly 
circular (Fig. 132) smooth, wet-shining, 
opalescent colonies (white by reflected 
light, pale brownish by direct trans- 
mitted light, bluish or pink-opalescent 
by oblique lighting). Although white 
at first by reflected light, the surface 
colonies become brown through the for- 
mation of a dark colored, water-soluble 



THE BROWN ROT OF SOLANACEAE : CAUSE 



183 




Fig. 126. — A. Bacterium solanacearum on red-fiowered Impatiens balspmina. 
Left-hand shoot inoculated July 2, 1918. An internal brown stain extended down 
the stem a foot or more beyond the needle pricks which were at A'. Height of 
plant 24 inches (to top of curve of wilted stem). Lower side-shoots still healthy. 
Photographed July 30, 1918, }i natural size. 

B. Stem of garden balsam from same series as A, but enlarged to show vascular 
black stripe visible through the unbroken translucent cortex. Plant inoculated 
20 days and about 4 inches above part here shown. X 4. 



184 



BACTERIAL DISEASES OF PLANTS 




Fig. 127. — Ricinus communis showing profound dwarfing due to Bacterium 
solanacearum. The left-hand plant was inoculated at the base of the hypocotyl 
during germination by three delicate needle pricks. Checks at right. Photo- 
graphed July 15, 1918. Time 19 days. Experiments of Smith and Godfrey. 



THE BROAVN ROT OF SOLANACEAE : CAUSE 



185 




Fk;. 128. — A, B. Vigorous Helianthus annuus (common sunflower) needle- 
pricked in the stem at X with Bacterium solanacearum plated from inoculated dis- 



186 



BACTERIAL DISEASES OF PLANTS 



pigment especially when Witte's peptone is used. On agar plates 
the young surface colonies are rather watery and will often flow 
if the plates are tilted to a vertical or semi- vertical position (Fig.. 
133). On gelatin plates the colonies are small and not charac- 
teristic. On steamed potato cylinders the bacterial slime is 
white at first, becoming gradually dark brown or black. It does 
not grow readily on raw potato (contrast with No. VII), Some 
of its most striking characters are: irregularly roundish, small, 



^ 







Fig. 129. — Cioss-sfction of a niature Florida potato attacked by Bacierium 
solanacearum showing brown stain and gray ooze in the vascular region. A 
natural infection bv way of the rhizome. 3? 



opalescent surface colonies on agar-poured plates, sensitiveness 
to dry air, bipolar staining, aerobism, liberation of brown stain 
in tissues of its hosts, dark stain on steamed potato and agar, slow 
clearing of milk without precipitation of the casein, bluing of 
cream-free litmus milk, non-liquefaction of gelatin (at least 
during the first few weeks), reduction of nitrates and failure 
to grow in Cohn's solution. 

It loses virulence rather quickly, dies out early on various 

eased vanilla, which was inoculated from tomato, which was inoculated from dis- 
eased Georgia Ricinus. Sub-cultures from single colonies on agar-poured plates 
were used in each case. Six plants (of which these were 1 and 2) were inoculated 
and all became diseased and badly dwarfed. See Fig. 131. Bacteria were abundant 
in the xjdem vessels of the stem and also in the root 2 inches below the crown (12 
inches below the needle pricks) in both plants. There was a brown stain in the 
vascular system. Time, 10 days. Plants inoculated July 12, 1918. Photographed 
July 22. }i natural size. 



THE BROWN ROT OF SOLANACEAE : CAUSE 



187 




Fig. 130. — Livid color and bacterial ooze on a petiole (p) of Tropoeolum majus 
(common garden nasturtium). Inoculated by needle pricks June 26, 1918, on 
the hypocotyl under the petiole with Bacterium solanacearum plated from an in- 
oculated wilting tomato plant. Photographed July 1. X 5. 



188 BACTERIAL DISEASES OF PLANTS 

media, and there are at least two strains: one of which splits 
fats (cream, etc.) with the formation of an acid (var. asiaticum 
EFS). The flagella are rather hard to stain (Fig. 134). It keeps 
best in milk, or litmus milk. 

Technic. — Because in the host plants the parasite is promptly 
followed by saprophytes which often supplant it, cultures are 
best made at some distance from the ground and out of parts 
recently diseased. It is a rather difficult organism for the 
beginner to isolate, owing to the fact that while it grows readily 
on agar-poured plates, the colonies during the first week re- 
semble those of various saprophytes, so that only after some 
days can they be picked out easily by the beginner, i.e., when the 
brown stain has developed, but then they are apt to have lost a 
portion of their virulence, or may be dead. The best way is to 
make transfers early from a whole series of numbered colonies 
that look hopeful, w^atch the plates for opalescence in surface 
colonies, and later discard all sub-cultures except those from 
colonies which have browned properly. The organism may be 
kept in agar, milk, sugared peptone water, etc., but transfers 
should be made as often as once every 2 or 3 weeks, and the 
tubes should be kept in a cool box. It is a good plan also to pass 
the organism frequently through susceptible plants that it may 
retain its virulence. 

For inoculation purposes select young rapidly growing shoots 
of nasturtium, tomato, potato, tobacco, balsam or sunflower. 
Inoculate by needle-pricks in various ways, i.e., on leaf -blades, 
petioles and soft stems, and, for contrast, into hard stems 
and well-developed leaves. Also drench a soil with cultures 
(young agar-streak suspensions in water), and plant in it 
tomatoes or Daturas with broken roots. Those which have 
stood in the seed-bed rather too long and are large may be 
used for this purpose. But if one has access to material from 
the field, diseased tobacco stems, or tomato stems may be used 
instead of cultures for infecting the soil, or along with cultures 
as an additional experiment, the diseased plants being buried 
in the soil a few days before the tomatoes or Daturas are 
transplanted. 

For successful inoculation the plants should not be too old, 



THE BROWN ROT OF SOLANACEAE : TECHXIC 



189 



and must continue to grow rapidly for at least several weeks. 
Only disappointment will result from inoculations on old slow- 
growing, woody plants, or with cultures which have been in the 
laboratory for a considerable period. The best results may 




Fig. 131. — Common sunflowers (Xos. 3 and 4) inoculated with Bacterium 
solanacearum bj" needle pricks on July 12, 1918, at X (on the stem) and badly 
dwarfed and wilting. The controls at either side were 33 inches high. Photo- 
graphed July 30, 1918, about }4 natural size. Experiments of Smith and Godfrey. 

be expected from cultures isolated the same summer they are 
used. 

Determine 

1. For the organism. Morphology. — Size in microns (stain 
with methyl violet or Ziehl's carbol fuchsin), form, aggregation 



190 



BACTERIAL DISEASES OF PLANTS 




Fig. 132.— Surface and buried colonies of Bacterium solnnacearum (Ricinus 
wilt) plated a second time from inoculated tomato. Agar plate poured July 6, 
1918. Photographed July 10, with oblique light. Shows characteristic irregu- 
larity of surface colonies. At X is a white concentric striate intruding colony. 
X 10. 



THE BROWN ROT OF SOLANACEAE : MORPHOLOGY 191 




Fig. 133.— Surface and buried colonies of Bacterium ,okmacearum. Agar 
pouredplateof July 13, 1918. Photographed July 17, 1918. X 8. Four of the 
fave surface colonies flowed when the plate was clamped vertically to photograph. 
b rom a broken negative. -^ i- & h 



192 



BACTERIAL DISEASES OF PLANTS 



of elements, chains, filaments, pseudozoogloeae, motility on mar- 
gin of a hanging drop (best seen by flooding a young agar- 
streak culture with sterile water and taking a drop from the 







' a 



.M 



Fig. 134. — Flagellate rods of Bacterium solanacearum: a, Medan III from 
Sumatra (Courtesy of J. A. Honing), h, North American organism (from North 
Carolina). Each X 1000. 




Fig. 135. — Stab cultures of Bacterium solanacearum after 5 days at 20°C. in 
+ 10 nutrient beef peptone gelatin. Organism isolated from Baltimore Tropaeo- 
lum. No liquefaction. 

top of the water after an hour or two) , presence and distribution 
of flagella which are hard to demonstrate (try van Ermengem's 
silver-nitrate method, Lowit's method, Pitfield's stain, etc.), 



THE BROWN ROT OF SOLANACEAE : MORPHOLOGY 193 

absence of endospores (heat, spore stains), bipolar staining (us- 
ing methylene blue), Gram's stain, acid-fast stain. Presence or 
absence of involution forms. 

Cultural Characters. — Behavior in nutrient agar (shape of 
surface colonies, color in various lightings) and in gelatin (thin- 
sown plates) ; streaks and stabs in agar and in gelatin (Fig. 135) ; 
growth and color on steamed potato (keep several weeks) ; 
behavior in bouillon; nitrate bouillon; Cohn's solution; Uschin- 



FiG. 136. — Cross-section of young Virginia potato luher diowing that starch 
has been removed from the vascular region occupied by Bacterium solanacearum . 
This was done by the potato plant,, not by the schizomycete. Surface unruptured . 
Tuber invaded through the vascular system of the rhizome. No fungi present. 

sky's solution; milk, which should be kept 10 weeks (how soon 
can you see your pencil, or read coarse print behind a tube of 
such milk?). Watch carefully to be sure that the clearing is not 
due to precipitation of the casein. When the milk has cleared 
to your satisfaction add some drops of strong hydrochloric acid. 
How do you explain the result? Behavior in lavender-colored, 
cream-free litmus milk (which should become and remain blue; 
watch closely to be sure that no acid is formed). Now add 
ammonia water, drop by drop, very gradually to check tubes of 



194 



BACTERIAL DISEASES OF PLANTS 



plain milk with shaking and compare the reaction with old milk 
cultures of Bacterium solanacearum.. Then neutralize, and some- 
what more, with hydrochloric acid and observe the second result. 
In view also of the bluing of litmus milk what do you conclude 
as to the probable cause of the slow clearing of the milk cultures? 
Distil old milk cultures and test steam for presence of ammonia. 
Test in peptone water in properly made fermentation tubes with 
various sugars and alcohols (compare with No. XII). Can you 




Fig. 137. — Longitudinal section through an inoculated potato stem showing 
the red stained dense mass of Bacterium solanacearum restricted to a single spiral 
vessel. X 170 circa. 

obtain growth in the closed end with any carbon food? (Read 
what is said in " Bacteria in Relation to Plant Diseases," Vol. I, 
pp. 53-54, respecting good and bad fermentation tubes). If you 
have time study the nitrogen nutrition of the organism. Can it 
use asparagin? Salts of ammonia? Try Meyer's mineral solu- 
tion: a, with ammonium citrate; b, with ammonium lactate. It 
should grow abundantly in a, and not at all in b. Can you find the 
cream-splitting form? The writer knows it only from Sumatra. 



THE BROWN ROT OF SOLANACEAE : ENVIRONMENT 195 

Non-nutritional Environment. — Effect of heat, of sunlight, 
of dry air (easily killed), of weak acids, of chloroform in bouillon, 
of salted bouillons? 

What is the maximum temperature for growth? the mini- 
mum temperature? the optimum? What is the effect of freez- 
ing? Why does the organism so readily lose virulence (power 
to infect) ? Can you discover any way to restore lost virulence? 
Any convenient way to hasten its loss? 

There is reason to think that it sometimes dies out of soils 
or is converted into an ordinary saprophyte (my own observa- 
tions and those of Coleman in Mysore). If so, we ought to 
be able to bring this about at will, thus disinfecting fields on 
which certain crops cannot now be grown profitably on account 
of its presence. Honing has found a soil-organism which is 
harmful to it. Can you find any organism which, when sown 
broadcast on a field, will destroy the parasite without injuring 
the host? Cultural studies may eventually suggest the proper 
treatment. 

For the disease. Signs. — Period of incubation. Time 
required to infect the whole plant. Relative effect of few vs. 
many punctures; of root vs. stem inoculations; of stem vs. leaf 
inoculations; of inoculations on young vs. old plants. The 
very susceptible tomato, Livingston's Dwarf Aristocrat, may 
be used for this purpose, inoculating first when 3 inches high 
and again when 2 feet high. On inoculated tomatoes how soon 
do the leaves begin to bend downward? Where first, and how 
soon, do the root-anlage appear on inoculated tomato shoots? 
Where do they naturally appear later on uninoculated plants? 
Can such root-anlage be made to develop further? Bind on 
wet sphagnum or bury a portion of the stem without separating 
it from the plant and examine from time to time. Have you 
found any plant in which the disease occurs without the brown 
stain? Are part of the signs due to a toxin? Inoculate 
seedling Ricinus by needle-pricks at top of hypocotyl as the 
plant emerges from the soil and compare subsequent growth 
with that of control plants: there is a conspicuous dwarfing 
due to such inoculations (Fig. 127). Consult also Fig. 131 and 
try Helianthus annuus. Read what Hutchinson says about 



196 



BACTERIAL DISEASES OF PLANTS 



toxins. You will find an abstract in "Bacteria in Relation to 
Plant Diseases," Vol. Ill, p. 267. 

Write a description of the disease, drawn (of course) from 
your own observations on either sunflower, tomato, potato, 
or tobacco, or all four. If you have time, make a special study 
of the dwarfing effects of the parasite. To what poison is this 




Fig. 138. — Cross-section of a potato stem in the vascular region showing 
vessels occupied by Bacterium, solanacearum and the beginnings of a bacterial 
cavity. Plant inoculated on a leaflet, May 27, 1895, and collected June 17. 
Stem sound externally. Block 113. X 800. 



due? Can you produce it with extracts of the bacteria: a by 
injection, b by watering the soil? 

Histology. — Section, stain and study leaves and stems for 
action of the organism on the tissues. Make good permanent 
preparations. Are vessels occupied first? Are cavities formed 
in the bundles? Have you found the organism in the phloem? 
Study invasion of the pith, of the bark. Draw what you see. 



THE BROWN ROT OF SOLANACEAE : HISTOLOGY 197 

How are the cavities produced? On the root-infected plants 
(tomatoes, Daturas, Tropaeolum, castor oil-plants), trace the 
bacteria from vessels in the broken roots into the stem and 
up the latter into the leaves. Are the unbroken roots sound? 
Also in stem-punctured plants follow the upward and downward 
movement of the bacteria. How far in advance of the brown 
stain can you trace the bacteria? Can you judge from the 
relative abundance of the bacteria in the vessels of the stem 
(above and below the point of inoculation) whether the infec- 




\ ^y^^ 



Fig. 139. — Empty and full (bacterially occluded) vessel in a potato plant. From 
same series of inoculations as Fig. 138. 

tion is moving up or down the stem? Can you trace the bac- 
teria along the vessels of the rhizome into the developing potato 
tuber? Is the starch in the tuber destroyed by the bacteria? 
Is it removed by the plant? Can you find any evidence of an 
attempt to ''cork out" the parasite? Study early stages of 
cavity formation, appearance of tyloses, and other effects of 
the organism on the tissues. 

Have you observed any bacterial motility in the plant? 
To see this you must ordinarily take parts only recently occupied. 



198 



BACTERIAL DISEASES OF PLANTS 



Does the organism come freely to the surface of the attacked 
plants (Contrast with No. V or XII) ? Does it freely attack the 
root-anlage (Contrast again with No. V)? Stomatal infection 
is easily obtained and studied on leaves of the Tropaeolum. 
Check Tropaeolums in the same pot are frequently attacked 
after some weeks. Consult Figs. 136 to 142. 

Variability. — How long does an attacked plant live? Have 
you seen indications of immunity on the part of inoculated 
plants? of recovery? Do you think rapid growth or great 
juiciness favors the progress of the disease? What results 







Fig. 140. — A detail from same series as Figs. 138, 139, showing the individual 

bacteria. X 1000 circa. 



did you obtain from your trials on young vs. old plants? Can 
you increase susceptibility by overwatering or decrease it by 
liming the soil? or by the liberal use of potash and phosphates? 
What do you conclude with reference to the effects of tempera- 
ture? Does the optimum temperature for the plant coincide 
with that of the micro-organism? Have you found any non-sus- 
ceptible varieties? A good non-susceptible tobacco would just 
now be worth its weight in gold! Why is the disease common in 
our Southern States and unknown or hard to find in our Northern 
States? Can you determine its presence in states north of 



THE BROWN ROT OF SOLANACEAE : YARI.ABILITY 



199 



Virginia and Texas? Specifically: is it in California, Maine, 
Wisconsin, Michigan, Ohio, Kentucky or New York? Does 
it occur in Canada? If not in these places, why not? 

Trcuisinission. — Have you seen any indication leading you to 
think that insects spread this disease? In 1896 I obtained 
very successful infections on potato, using the Colorado potato 
beetle {Leptinotarsa decemlineata) . Many narrow, dark, bacteri- 
ally infested streaks started in the bitten places and passed 




fe 






•\..- 



^?J 



,XJ^m 



-s^^^cm 





^■i^^ 



'/ 



^ 



Fig. 141. — Photomicrograph showing origin and structure of two incipient 
roots in an inoculated diseased tomato stem. Bacterium solanacearum occurs in 
some of the vessels at the lower left side. What stimulus sets the roots growing? 



rapidly down the stems, both stems and tubers being de- 
stroyed. If you have opportunity watch infected fields closely 
and if you obtain clues make some experiments. Hunger in 
Java incriminated several insects and also thought Phytoph- 
thora nicotiana paved the way for this parasite. Are plants on 
wet soils more liable to it? Do the roots of the infected plants 
usually bear nematode galls? Are plants on limestone soils 
free from it? 

Of course, one susceptible crop should not closely follow 



200 BACTERIAL DISEASES OF PLANTS 

another. There should be a long rotation on infected lands, 
using non-susceptible species — clovers (?), grasses, etc. 

In this connection it is very important to know whether 
any of our common forage crops are susceptible and also whether 
many of our American weeds are subject to this disease, and 
might act as hold-over hosts. Honing found susceptible weeds 
in Sumatra; Stanford and Wolf have found them in North 
Carolina. Long ago I found it readily inoculable into Datura 
stramonium (the jimson-weed). Has anyone found it naturally 




Fig. 142. — Tyloses in vessels of a potato stem attacked by Bacterium solanace- 
arum. At X is a vessel occupied by the bacteria. 

on this plant? In this connection read Stanford and Wolf's 
papers. The disease has been reported to me several times from 
Florida as occurring on ''new land." 

LITERATURE 

For literature, etc., consult Van Breda de Haan's Wilt of 
Peanut; Brown Rot of Solanaceae; and Wilt Diseases of Tobacco 
in "Bacteria in Relation to Plant Diseases," Vol. Ill, pp. 151- 
153, 174-219, and 220-271, Carnegie Institution of Washington, 
1914. Ihid., Vol. I, plates 4, 24, 25, 26, 27, and Fig. 10; and Vol. 
II, Fig. 1. 



THE BROWN ROT OF SOLANACEAE : LITERATURE 201 

Stanford, E. E. Studies on Resistance of Tomatoes to 
Bacterial Wilt. X. C. Ag. Exp. Sta. 40th Ann. Kept., 1916- 
1917, pp. 92-93. 

See also Stanford, E. E. and Wolf, F. A. ''Studies on Bac- 
terium solanacearu7n.'" Phytopathology, Vol. VII, No. 3, June, 
1917, pp. 155-165. 1 Fig. 

The first note on this disease was by Prof. T. J. Burrill 
in 1890. The first paper relating the disease to a definite micro- 
organism was by the writer in 1896: ''A Bacterial Disease of 
the Tomato, Eggplant, and Irish Potato." U. S. Dept. Agric. 
Bull. No. 12, Div. Veg. Phys. and Path., Washington Govt. 
Printing Office. Here the name Bacillus solanacearum first 
appears. 

The first paper proving the disease to occur in tobacco was 
also by the writer: "The Granville Tobacco Wilt." U. S. 
Dept. Agric, Bu. PL Ind., Bull. 141, part II, Washington Govt. 
Printing Oflace, 1908. 

The last notes are by 

Smith and Godfrey, Brown Rot of Solanaceae on Ricinus, 
Science, N. S., Vol. XLVIII, July 12, 1918, pp. 42-43; and by 

Smith and McCulloch, Bacterium solanacearum in Beans, 
Science, N. S., Vol. L, Sept. 5, 1919, p. 238. 



V. BACTERIAL CANKER OF TOMATO 

Type. — ^This disease (Figs. 143 and 144), which for want 
of a better name I first called The Grand Rapids disease, after 
the locality in Michigan from which it was first sent to me and 
where it occurred seriously over large fields, is an infectious pai- 
enchymo-vascular wilt of the tomato (and probably also of the 
potato), somewhat resembling the brown rot of Solanaceae due to 
Bacterium solanacearum and often confused with it, but differ- 
ing in a number of particulars, e.g., it is highly infectious through 
the above-ground parts ; there is less brown stain in the bundles 
and not so strong a tendency to develop incipient aerial roots 
(Fig. 145) ; there is a slow shriveling of the leaflets one after 
another (Fig. 146) rather than a sudden general wilt of the leaf; 
the petioles are not reflexed; the meristem is attacked and cor- 
roded into cavities, e.g., the heart of the incipient roots (Fig. 
147); the phloem is specially susceptible to disorganization 
(Figs. 148 to 150) ; and there is a strong tendency of the bacteria 
to come to the surface through fissures on the shriveling leaves, 
fruits and shoots (Figs. 151 to 154), thus affording an abundant 
surface slime for the above-ground infection of neighboring 
plants (through stomata) ; infection through broken roots has 
also been observed (Fig. 155). The disease spreads easily from 
one plant to another often by stomatal infection (Figs. 156 to 
158) and is very destructive. It is, I believe, primarily a 
phloem disease. I think also that it is a seed-borne infection. 
I have seen its yellow slime close under the seeds in the middle 
of green tomato fruits, both in the vascular bundles of the peri- 
carp and in those of the placenta, and also once in the base of 
an immature seed, but I have not yet actually traced it into or 
plated it from the ripened seeds. Whether or not it actually 
occurs in the interior of seeds capable of germination, the fre- 
quent extensive invasion of the outer part of the tomato fruit 
is certain to bring about a surface contamination of the seeds. 
It occurs in the Northern United States both under glass and 

202 



BACTERIAL CANKER OF TOMATO: TYPE 



203 




Fig. 143.— Tomato plant inoculated 2]4, months with a pure culture of the 
non-motile, yellow Aplanohncter michiganense plated from a New York tomato, 
showing slow, irregular wilting of the leaflets. Photographed Nov. 25, 1912. 
This and Fig 144, made in 1915, may be compared with pure culture inocula- 
tions of 1909 shown on plates 12 to 15 "Bacteria in Relation to Plant Diseases," 
Vol. III. 



204 



BACTERIAL DISEASES OF PLANTS 




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BACTERIAL CANKER OF TOMATO: TYPE 



205 



in the field. I have had it from Michigan, Western New York 
and Eastern Massachusetts. So far it has not been reported 
from the Southern States, but I beUeve I had it once from 
Texas without recognizing it as distinct from the brown rot. 
It undoubtedly occurs in Europe. It should be looked for in 
England, France, Belgium, Germany and Italy. 




Fig. 145. — Stems of tomato plants inoculated with A planobacier michiganense 
October 5, 1909, and photographed January 17, 1910, when most of the foliage 
had shriveled. These show slight tendency to formation of adventitious roots, 
as compared with those attacked by Bacterium solanacearum. Compare with 
Fig. 122 inoculated onlv 12 days. 



Cause. — This disease is due to Aplanobacter michiganense 
EFS. This is a rather short, viscid, yellow, non-motile, non- 
sporiferous, non-gas-forming, aerobic, very slowly liquefying, 
non-nitrate-reducing, rod-shaped schizomycete (Fig. 159) form- 
ing slowly on beef-peptone agar-poured plates small circular 



206 



BACTERIAL DISEASES OF PLANTS 



colonies which become darker with age but are always yellow 
(not orange colored). Katherine Bryan has also isolated a 
form which spreads on agar. It stains by Gram, but is not acid- 
fast. It grows copiously in milk with slow coagulation,! form- 
ing a thick pellicle and a broad yellow rim. A very little acid 
seems to be produced, as litmus in milk becomes gray or purplish 




Fig. 146. — Tomato leaf showing irregular wilting of leaflets due to Aplano- 
bacter michiganense. Stem inoculation by Mary Katherine Bryan using a round 
colony. Photographed May 3. 1917. 

red before it is reduced. It grows well on cylinders of steamed 
potato standing in water, the bacterial layer being pale yellow 
at first, becoming bright yellow (R2, light cadmium), smooth and 
inclined to spread. In color, the slime of this organism_on 
potato is not unlike that of Bacterium, campestre, but the grov.^th 
is less prolonged. The substratum out of the water is grayed. 
Its growth in peptone bouillon, and on gelatin- and agar-poured 



BACTERIAL CANKER OF TOMATO! CAUSE 207 

plates is very slow, often so slow as to discourage isolations. It 
is sensitive to heat, acids, even of beef juice (Fig. 160), and sodium 
chlorid. It endures drying well. It gradually loses virulence 
on culture media. Litmus agar containing dextrose is reddened. 
It does not grow in Cohn's solution or in Uschinsky's solution. 
The slime is apt to be viscid, both on potato and on agar. It is 
also viscid in certain sugar solutions. Is it ever viscid in milk? 
Spieckermann in Germany has described a potato disease 
of slow development but considerable importance, due to a slow- 
growing, yellow, non-motile schizomycete which I suspect to be 
this organism, or one closely related to it. This he has named 
Bacterium sepedonicum (A pi. sepedonicum in my terminology.) 




Fig. 147. — Tomato stem in cross-section sliowing the center of an incipient root 
honeycombed and destroyed by Aplanobader michiganense. 

Technic. — Because of the slow development on poured plates, 
isolations from the plant may be made in deep Petri dishes by 
parallel streaks on slices of steamed potato, where the last 
wipings of the needle (there should be 6 or 8) should yield single 
colonies from which poured plates and sub-cultures may be made. 

The upper part of stems of young rapidly growing plants 
may be used for inoculations which may be by needle-pricks from 
young potato cultures. If the organism is virulent, results will 
begin to appear in 10 or 15 days, but the inoculated plants should 



208 



BACTERIAL DISEASES OF PLANTS 



,'. >^- z::h.^ 






■V:/; 



.-::/ , ^■>',r^^- '•; 










b:--!.. 







Fig. 148. — A. fi. Tomato stem inoculated with Aplanobacier michiganense, 
showing bacterial cavities to either side of the wood, i.e., cavities originating in 
the phloem; xylem not much disturbed. A later stage than Fig. 143. The two 
views are on opposite sides of the same stem and at the same level. 



BACTERIAL CANKER OF TOMATO: TECHNIC 



209 





Fig. 149. — Longitudinal .section of a tomato .stem showing the phloem attacked by 
Aphinobacter michiganense and the other tissues free. 



\ 



i 




Fig. 150.— Cross-section of a tomato stem showing a small group of sieve- 
tubes attacked by Aplanobacter michiganense and the surrounding tissue free 
Sieve-plates are visible. 



210 



BACTERIAL DISEASES OF PLANTS 




Fig. 151.— (1) Green tomato fruit sound e^fternally but showing the pedicel 
(at x) honeycombed and full of Aplanobader michiganense. At y also there is a 



BACTERIAL CANKER OF TOMATO: TECHNIC 



211 




%■ 



^ 




Fig. 152. Fig. 153. 

Fig. 152. — Tomato stems showing loss of color (whitening) and swelling 
prior to the formation of longitudinal cankers. Plants inoculated for several weeks 
below parts here shown, with Aplanobader michiganense (round colony). Photo- 
graphed May 3, 1917. Natural size. 

Fig. 153. — Typical canker-crack on a tomato stem due to Aplanobader 
michiganense. Plant inocidated on the stem below the part here shown. 



natural crack or canker from which the yellow bacteria are oozing abundantly. 
X 5. 

(2). Another green fruit from which the diseased pedicel has been removed. 
On the darker parts of the scar are masses of the j-ellow bacteria which have pene- 
trated into the interior of the fruit which is sound externally. X 8. 

Natural infections from a hothouse in Massachusetts. Received in September, 
1915. Theh-e yielded a long series of pure culture inoculations. 



212 



BACTERIAL DISEASES OF PLANTS 



or more. 



The 



10' 



be kept under observation for three months 

spreading colony is the more virulent one. 

Agar-plate cultures, especially when 
' made directly from the plant, should be 

kept under observation for at least fifteen 
days. Colonies are seldom ready for ex- 
amination earlier than the eighth day. 
Agar titrating +8 or +10 on Fuller's scale 
is better for plate cultures than + 15 or 
that which is more acid. In general, in- 
oculations will be more successful if made 
with sub-cultures from recent isolations 
rather than with those long in the labora- 
tory. They should be of the same sum- 
mer, if possible. 



/' 



Fig. 154. — Green 
tomato fruit oozing 
Aplanobacler michigan- 
ense around the pedi- 
cel. The result of a 
stem inoculation. 
Photographed from al- 
coholic material. 



Determine 



For the organism. Morphology. — 
Size in microns, form, aggregation of elements. Examine for 
motihty, stain with flagella stains, search for endospores, stain 




Fig. 155. — Tomato plant infected with a pure culture of Aplanobacter michi- 
ganense through the soil by way of broken roots. Inoculated Nov. 17, 1910. 
Photographed Dec. 21, 1910. This also shows irregular wilt of leaflets. 

viscid cultures for a capsule; try Gram's stain; acid-fast stain. 
Any evidence of involution forms? 



CANKER OF TOMATO: CULTURAL CHARACTERS 



213 



Cultural Characters. — Growth on thick vs. thin-sown agar 
and gelatin plates. Behavior in agar and gelatin streaks and 
stabs. Can you find two types of colony: a round, b spreading? 
On which agar do you get the best results (a) +15, (5) +6, or (c) 
+ ? Will the organism grow on — 1 agar ? — 20 agar ? Appearance 
on steamed potato; behavior in bouillon, and in nitrate bouillon; 




Fig. 156. — Stomatal infection on sprayed tomato leaf due to Aplanobacter 
michiganense. The stoma is at A'. Under it the cells are destroyed and the 
bacteria are abundant in the bundle. Time, 21 daj^s. 



behavior in milk and litmus milk; streaks on litmus-lactose agar; 
growth in peptone w^ater in fermentation tubes with various sugars 
and alcohols. Relative rapidity of growth in diluted tomato 
and potato juices, as compared with Dunham's solution and pep- 
tone bouillon (Titrate each to determine acidity and inoculate 
sparingly with a 1-mm. loop from a fluid culture) ; behavior in 



214 



BACTERIAL DISEASES OF PLANTS 



various synthetic media; Cohn's solution, Uschinsky's solution, 
Fermi's solution, Meyer's solution, etc. 

N 071-nutrithonal Environment. — This organism offers a number 
of very interesting problems. Perhaps you can help to settle 
some of them. Can you get any growth in milk, or in bouillon, 
at 1°C., orat37°C.? What is the thermal death-point? Deter- 
mine its toleration for malic, citric, and tartaric acids begin- 




FiG. 157. — Longitudinal section of a tomato leaf showing bundle disorganiza- 
tion due to Aplanohader michiganense. Stained section from a sprayed leaf. 
Time, 21 days. 

ning with -1-20 (the acid of the tomato is said to be tartaric) ; for 
sodium hydrate beginning with —20°. My own experiments 
lead me to think it is rather tolerant of alkali and compara- 
tively sensitive to organic acids. Try salted bouillons — 1, 2, 3, 
4, 5, etc., per cent. Determine if you can the inhibiting sub- 
stance in the culture-media in which it makes such a slow growth; 



CANKER OF TOMATO: NON-NUTRITIONAL ENVIRONMENT 215 



is it the peptone? or the sarco-lactic acid of the beef juice? 
Test its growth in diluted steamed tomato juice or potato 
juice, and in the same with very small quantities of lactic 
acid added (the first days are the important ones for observa- 
tion). What are your conclusions? Can you devise an agar 
medium on which it wdll grow readily? Determine effect of 
drying, of insolation, of germicides. What causes loss of 
virulence? What is the best medium for its retention? 

For THE DISEASE. Signs. — Period of incubation. Time 
between appearance of the disease on the inoculated leaf and gen- 
eral infection of the plant. 
What are the most conspi- 
cuous external indications of 
his disease? Why are the 
leaves not reflexed as in case 
of No. IV? Note especially 
the swollen whitish lines 
followed by corrosion and 
cracking open of stems, pet- 
ioles, etc., and the irregular 
wilting of the leaflets on at- 
tacked leaves. Write a de- 
scription of it. 

Histology . — Cut free- 
hand sections of leaves, 
stems, roots and fruits, and 
determine: a, extent of 
movement of the bacteria in the plant; 6, tissues attacked 
(xylem, phloem, parenchyma) ; c, movement of the bacteria to 
the surface of the plant; d, evidence of bacterial entrance through 
stomata. Make stained preparations from paraffin-embedded 
material, showing the bacteria in these various tissues (stain 
with Ziehl's carbol fuchsin). How are the cavities formed? Is 
cellulose destroyed? 

Variability. — How long does an attacked plant live? Are 
young plants more susceptible than half-grown or old plants? 
Search in the field and hothouse for resistant varieties, and if 
you see indications of resistance, make experiments. If you 




Fig. 1.58. — ^Detail showing infection 
of a small bundle in a tomato leaf sprayed 
with Aplanobacter michiganense. Time, 21 
davs. 



216 



BACTERIAL DISEASES OF PLANTS 



I ^ 



/ 



have time and a place include a variety test in your inoculations. 
Determine if the shoots of the potato are susceptible (do not 
expect rapid infection) ; look for it in the field upon potatoes : 
(a) stems; (b) tubers. (Read Spieckermann's papers.) 

Transmission. — Determine whether I am right in thinking 
that the organism freely oozes to the surface of diseased tomato 

plants, and that infections are 
['/ ** *^''' commonlyabove ground 

; - ' * ' . * through stomata. 

It is very important to 
know whether it is borne on 
tomato seeds (I believe it is, 
since it resists drying and we 
find it very commonly in the 
fruits). If so, it may come 
into the field from the seed 
bed, as in case of Bacterium 
campestre (No. II). To estab- 
lish this conclusively, I have 
done it only inferentially, 
would be a fine practical con- 
tribution to our knowledge of 
the disease. 

Once in the field, how is 
the organism carried from 
plant to plant? Rains ex- 
cluded, it can scarcely be wind- 
borne, do you think? If you 
have opportunity to study this 
disease in the field (the writer 
has not had) you should ex- 
amine particularly for carriers of infection (insects, etc.), for 
evidence of under-ground infection, i.e., through the root-system, 
and for transmission on seeds taken from diseased plants. The 
disease was so prevalent and destructive at Grand Rapids, 
Michigan, that seemingly it must have begun early in the life 
of the plants. 

It escaped from my control in one of the Department of 



^'-•'^ - 



{ 



Fig. 159. — ^Rods of Aplanobacter 
tnichiganense. From an agar culture 2 
daj's old, stained by van Ermengem's 
silver nitrate method. X 1000. 



BACTERIAL CANKER OF TOMATO: TRANSMISSION 217 



Fig. 160. — Aplanobader michiganense: uniformly sown on +15 peptone-beef 
agar-poured plate, showing growth of colonies inhibited everywhere except in 
the vicinity of the central white intruding colony which is an alkali-producing 
schizomycete. At x there is an inhibiting mold colony, probably an acid producer. 
There was a brown stain in the agar between and around the yellow colonies. 
The colonies on the inner part of the periphery were fluorescent. Those on the 
outer one-fourth were not fluorescent. Photographed December 13, 1915. 
Slightly enlarged. 



218 



BACTERIAL DISEASES OF PLANTS 




Fig. 16L— The Berkshire, Mass., potato disease (net-necrosis). SHce of tuber 
photographed by reflected light. February 28, 1919. X 4. 



BACTERIAL CANKER OF TOMATO: TRANSMISSION 219 

Agriculture hothouses and infected various clieck tomato plants 
and also a bed of West Indian plants {Solanum mammosum) 
said to be resistant to Bacterium solanacearum. This unwelcome 
infection was attributed to spatterings from the gardener's 
hose. I first called attention to this method of dissemination 
in 1914. The experiences of growers in New York and Massa- 
chusetts show that it is capable of doing much damage to hot- 
house tomatoes. 

Host Plants. — It is very important to determine whether 
this parasite has other hosts than the tomato. I believe it 
occurs also on the potato but the evidence is not yet conclusive. 

In the wdnter of 1918-19 I received potatoes from Berk- 
shire Co., Mass., said to be fair samples of a great many occurring 
in that locality. These tubers were sound externally but the 
outer one-half inch or more of their flesh was mottled with 
numerous brown spots, forked lines and streaks (Fig. 161). On 
studying sections under the microscope, no distinct lesions 
were observed but foci of bacteria were found in the center of 
some of the spots. In all the tubers I examined, the stem end of 
the tuber was always badly diseased, but often the eye-end 
was free (Fig. 162). When the flesh of the tuber was examined 
in thin section (1-3 mm.) by transmitted light, the brown spots 
and streaks were seen to be surrounded by a narrow clear zone 
indicating disappearance of the starch in the surrounding 
tissues (Fig. 163) which was confirmed by tests with iodine. 
Several organisms were cultivated out on potato. Some made 
a pale whitish slime at first, becoming distinctly yellow, but in 
other cases pure white cultures were obtained, and in many 
instances nothing whatever. Inoculation tests on tomato 
and potato gave nothing definite. 

This disease was not discovered by the planters in the field 
but during the winter in the stored tubers (variety, Green 
Mountain). This new disease has received the name of "Net- 
necrosis." By some it has been ascribed to frost injuries, but 
I cannot think the phenomena as it occurred in Massachusetts 
in the winter of 1918-1919, and as shown on the accompany- 
ing plates, w^as due to freezing. When the plates were made 
I believed the disease due, probably, to bacteria, but now I 



220 



I3ACTEEIAL DISEASES OF PLANTS 




Fig. 162. — Cross-sections of a Green Mountain potato tuber from Erwin 
E. Maynard, Savoy Center, Berkshire Co., Mass., showing net-necrosis. Stem- 
end diseased; eye-end free. Photographed April .3, 1919. 



BACTERIAL CANKER OF TOMATO: HOST-PLANTS 221 



Fig. 163. — Like Fig. 161, but from a thin section photographed by trans- 
mitted Hght to show narrow clear spaces (starch. destruction) around the browned 
vascular Inindles. X 4. 



222 BACTERIAL DISEASES OF PLANTS 

have no definite opinion as to its cause. Possibly it is of fun- 
gus origin. The disease appeared again the following year in 
Berkshire County, according to Mr. Maynard, but less seri- 
ously. Such tubers give spindling plants. My illustrations 
should be compared with those of Jones, Miller and Bailey in 
"Frost Necrosis of Potato Tubers" (Agr. Exp. Sta. of Univ. of 
Wis. Research Bui. 46, Oct., 1919) which appeared since the 
above was in type. 

LITERATURE 

The first paper definitely relating this tomato disease to a 
particular organism was by the \vriter in 1910, Science, N. S., 
May 20. 

For literature, etc., consult The Grand Rapids Tomato 
Disease in " Bacteria in Relation to Plant Diseases," vol. Ill, 
pp. 161-165. In this connection, read also what is said con- 
cerning Spieckermann's potato disease, Ibid., pp. 166-167. 

Spieckermann and Kotthoff's full paper on the ring rot 
of the potato is in Landw. Jahrhucher. Bd. 46, Heft 5, 1914. 

See also Paine, Sydney G. and Bewley, W. F. ''Comparison 
of the Stripe Disease with the Grand Rapids Tomato Disease" 
in Studies in Bacteriosis. IV. — "Stripe" Disease of Tomato. 
The Annals of Applied Biology, Vol. 6, 1919, Nos. 2 and 3, pp. 
200-202. 



VI. JONES' SOFT ROT OF CARROT, ETC. 

Type. — This is a rapid bacterial wet-rot of storage paren- 
chyma (roots, rhizomes, fruits and fleshy stems). It seldom 
attacks well-developed green parts, nor does it develop vigor- 
ously in storage tissues unless they are turgid. It was described 
in 1901 by Prof. L. R. Jones from carrot roots grown in Vermont, 
but he obtained it on the fleshy parts of many other plants by 
pure-culture inoculation, and it is now known to be widespread 
in nature on a variety of hosts. Probably it occurs all over the 
w^orld but its geographical distribution remains to be worked 
out. 

We owe most of our knowledge of this disease to Prof. 
Jones, but others have also studied it critically in recent years, 
notably, Harding and Morse, and to some extent also the author 
of this book. 

Since the appearance of Jones' first paper the same organism 
has been isolated from soft rots on other plants by several plant 
pathologists, who have studied and described it under other 
names. Furthermore, several other very closely related if not 
exactly identical soft-rot organisms have been discovered, de- 
scribed and named. M. C. Potter's white rot of turnips is due 
to this organism, and his paper appeared in 1899 (two years earlier 
than Jones' paper) , but he described as its cause a polar flagellate 
organism and his name, therefore, cannot be substituted. 

The parasitic action of all of these morphologically and cul- 
turally similar soft-rot schizomycetes is essentially the same and 
Harding and Morse believe all of them to be one species but I 
am not entirely committed to this belief. They enter the plant 
through wounds and rapidly disintegrate the susceptible parts 
into a soft, wet pulp (Figs. 164 to 166), having first poisoned the 
tissues by means of their by-products. They advance into the 
weakened tissues by way of the intercellular spaces and separate 

223 



224 



BACTERIAL DISEASES OF PLANTS 



the cells one from another by dissolving the middle part of the 
cell- wall, which is of a different composition from the outer part. 
The protoplasm of such separated cells is collapsed and dead, but 
the bacteria are not found inside the cells except in late stages 
of the disease. 

The first indications of disease in a carrot root are the ap- 
pearance of water-soaked (translucent) places around the in- 
fected wounds. These spots are visible in from 18 to 36 hours 




Fig. 164. — Bacillus carotovorus L. R. Jones, streaked for 3 days on raw carrot. 
Kept on the table at 23°C. in a large covered dry culture dish. The inoculation 
was from a rotting raw potato which was inoculated from a gelatin colony. The 
carrot was first washed, then soaked in 1:1000 mercuric chlorid water, and cut 
with a cold sterile knife. The left (check) part remained sound. 



after inoculation when the roots are held at 20° to 24°C. With- 
in two or three days this tissue breaks down, shrivels and exudes 
drops of a gray fluid swarming with the bacillus. Sometimes a 
thin, gray bacterial film also covers the surface. When a 2- 
mm. loop of a bouillon culture is placed on a slice of carrot in a 
covered Petri dish the water-soaked appearance may sometimes 
be seen in 6 hours at 20° to 23°C. In nature the rot usually 



JONES SOFT ROT OF CARROT, ETC.: TYPE 



225 



begins at the crown or at the root-tip. The disease continues 
in the stored carrots which often decay very rapidly and in large 
numbers. The core of the carrot rots more rapidly than the outer 
part of the root, and flabby roots are much less susceptible than 
turgid ones (Fig. 167). The attacked roots of half-long orange 
carrots are stained a dark brown, this color commencing within 
24 hours; those of the long-orange carrot are not stained or only 
slowly and slightly stained. Inoculated parsnip roots are 




Fig. 165. — Same as inoculated half of Fig. 164, hut after it had lieen dropped. 
Tissue entirely soft rotted e.xcept a tliin external layer. ^2 "'^t. size. 



changed to a clay color deepening to cinnamon brown. The 
spots on green tomato fruits are turned dark. Jones observed 
no stain in other inoculated rotting plants. Decay of the cruci- 
ferous roots was accompanied by an offensive odor. Decaying 
onions also emitted a bad odor. 

The disease has been seen in the United States occurring 
naturally or has been obtained artificially by pure- culture in- 
oculations on the following plants: carrot, parsnip, celery, 



226 



BACTERIAL DISEASES OF PLANTS 



lettuce, cabbage, cauliflower, turnips, radish, cucumber, musk- 
melon, potato, tomato and pepper (ripe and green fruits — 
faster in the latter), eggplant (ripe fruits), hyacinth (leaf and 
scape), and onion (bulb and leaf). 

Jones' inoculations failed on ripe oranges, bananas, pears 
and apples; on cauliflower, sweet potato, beet, asparagus; and 




Fig. 166. — Photomicrograph .showing separation of cells of carrot due to the 
action of Bacillus carotovorus. Inoculated from a beef-bouillon culture. Time, 
2 days. 1915. Organism 3a, in the laboratory several years. 



repeatedly on Irish potato tubers (once successful, however). 
They also failed on young carrot, parsnip and lettuce plants, 
and on the petioles and stems of the tomato. Most of his early 
inoculations were made in my laboratory in Washington early 
in the year (February to April) on roots and fruits from the mar- 



JONES' SOFT ROT OF CARROT, ETC.: TYPE 



227 




Fig. 167.— .4. Shows photograph cjf two carrots inoculated at the 



same time. 



228 



BACTERIAL DISEASES OF PLANTS 




,r%- 



■'\ 




/ 




Fig. 168. Fig. 169. 

Fig. 168.^ — Surface and side view of a raw potato tuber (Green Mountain) 
streaked 4 days, at 23°C., with Bacillus carotovorus from a gelatin colony (3a, 
long in my laboratory). The right-hand figure shows the depth to which the rot 
has penetrated. Photographed Jan. 16, 1916. 

Fig. 169. — Potato plant, variety Green Mountain, one shoot of which has 
been inoculated 7 days with Bacillus carotovorus. Inoculated shoot dwarfed 
and wilting with internal brown streaks. Organism less active than Bacillus 
phytophthorus. Photographed Feb, 8, 1915. 

from same (4-day) bouillon culture of Bacillus carotovorus. No. 1 was flabby, No. 2 
was turgid. Left check omitted. Time, 3 days. 

B. Same as A, but at the end of 6 days at room temperature (25°C.) in a large 
culture-dish. The inoculated half of the flabby carrot now shows a slight rot 
at the top where an unusually large amount of the cloudy bacterial fluid was 
deposited, i.e., much more than on the badly rotted piece. Experiment of May, 
191.5. Four days later there was little change. Right check omitted. This was 
still sound. 



JONES' SOFT ROT OF CARROT, ETC.: TYPE 229 

ket. Time of year, abi^ence of turgor, varieties used, or gradual 
loss of virulence on the part of the organism might make a differ- 
ence with potato tubers. Or it may be that what is here de- 
scribed as BaciUus carotovorus is a composite of two or more 
species. His conclusion at that time was that it did not attack 
the potato. 

According to my observations the organism does not lose 
virulence readily, and the culture I have (which came originally 




Fig. 170. — Green cucumber inoculated b}' longitudinal stabs introducing 
BaciUus carotovorus from gelatin colonies. Sliced and photographed January 
20, 1915, i.e., after days at 23°C. Interior soft rotted. Sliced (check) cucumber 
from the same lot on the right side, entirely sound. 3'2 J^^t. size. 

from Jones, but has been transferred many times in my labora- 
tory^) rots raw potato tubers readily (Fig. 168) and also attacks 
the soft green stems of this plant (Fig. 169). Its disintegrating 
action on many tissues other than those of the carrot, e.g., 
green cucumber fruits (Fig. 170), is very rapid. I have also 
obtained with it a calla lily rot resembling Townsend's rot 
(Figs. 171 to 173) and a rot of young leaves of carrot. 



230 



BACTERIAL DISEASES OF PLANTS 




Fig. 171.— Calla lily rot due to 
needle-pricks introducing Bacillus caro- 
tovorus. Leaf-stalk inoculated 48 
hours at A'. The rot extended intern- 
ally beyond Y. Another leaf-stalk of 
the same series rotted entirely across 
and fell over the third dav. 



In potato tubers a protec- 
tive layer of cork is often devel- 
oped under the rotting area (Fig. 
174). In the shoots of potato it 
is not partial to the vascular 
system but nevertheless may 
sometimes be found in vessels 
at a considerable distance above 
the place of inoculation (Fig, 
175). 

Cause. — The cause of this dis- 
ease is Bacillus carotovorus L. R. 
Jones. This is a gray- white, ^ 
non-capsulate, non-sporiferous, 
actively motile (2 to 5 flagel- 
late), peritrichiate (Fig. 176), 
slowly liquefying (gelatin, but 
not coagulated egg albumen or 
Loffler's solidified blood serum), 
nitrate reducing, milk curdling 
(by an acid), aerobic and facul- 
tative anaerobic, gas-forming 
(with muscle sugar, dextrose, 
saccharose, lactose and mannit 
but not with glycerin nor with 

' Wormald says yellow on Soyka's 
milk rice (.1 c). The quality of white- 
ness is variable as is that of any other 
color. I agree with Wormald that it is 
yellowish in contrast with the pure white 
of the rice medium, aliout as yellow as 
steamed potato cylinders, but I would 
call these white rather than yellow or 
more exactly following Ro nearly pale 
cream color, becoming cream color in old 
cultures. It is a matter of opinion. 
Very few white organisms are as white as 
white rice but nothing would be gained 
by calling all of them chromogens. For 
preparation of this useful medium see 
Eyre's Bacteriological Technique. 



JONES SOFT ROT OF CARROT, ETC. : CAUSE 



231 



potato juice in fermentation tubes, ^ the gas being 20 per 
cent COo and 80 per cent explosive), heat-sensitive (thermal 
death-point 51°C. — even 10 minutes in bouillon at 47°C. re- 
tards growth), dry-air-sensitive (sometimes even to 2 minutes' 
exposure — Jones), sunlight-sensitive (10 minutes of direct sun- 
light and 2 hours of difTused sunlight, fatal — Jones), not ex- 
ceedingly frost-sensitive (frozen in -f 15 peptone beef bouillon, 
in 1919, 13 per cent survived) short, rod-shaped, catenulate, 
or filamentous (up to 200^ or more — Jones) schizomycete 




Fig. 172. — Cross-section of Fig. 171 at Y, moderately enlarged to show char- 
acter of the bacterial rot. The tissue of the attacked part X did not hold the stain. 



(greatest observed variation in diameter 0.6 to 0.9^, usual 
diameter 0.7 to 0.8m), growing on the surface of agar-poured 
plates in the form of round, raised, smooth, gray-white, wet- 
shining colonies (2 days) having entire well-defined margins 
and a transient flaky areolation (XlO) with a slight fluo- 
rescence, the buried colonies being globose, oblong or spindle- 
shaped with irregular margins (X125), but, if thin sown, 

1 Repeated in 1919 in potato juice in fermentation tubes with contradictory 
results, using Jones 3a (branched) and also So received from Wisconsin in 1920. 
The latter sometimes gives a little gas and at othertimes not. 



232 BACTERIAL DISEASES OF PLANTS 

the surface colonies (Jones 3a, stock long in my laboratory) are 
sometimes more or less irregular in outline and may send out 
finger-like branched projections (Fig, 177). Fearing that in 
some transfer the labels of Bacillus aroideae and Bacillus caro- 
tovorus might have been interchanged I sent to Wisconsin in 
1920 for another culture of 3a and this in agar-poured plates 




Fig. 173. — Detail from Pig. 172 at A'. Much enlarged to show the bacteria dis- 
integrating the swollen cell-wall and confined to the interceUular spaces. 

gives round colonies (Fig. 178). The two stocks also differ in 
amount of gas formed from potato juice (Fig. 179) and in other 
ways. 

On the +10 gelatin-poured plates the margins of the young 
surface colonies (X125) are thickly set with parallel filamentous 
outgrowths (Figs. 180, 181), this fimbriate margin being about 
50m wide on the second or third daj^ The buried colonies 



JONES SOFT ROT OF CARROT, ETC. : CAUSE 



233 




Fig. 174. — Crot^s-soction of a McCijriiiick potato tul)er taken 12 days alter 
inoculation, i.e., when the rot had subsided, showing (at A'-A') the formation of an 
iniiibiting hiyer of cork under the rotted area. This tuber was streaked with 
Bacillus caroloL'orus at tlie same time as Fig. 168, and rotted as well at the begin- 
ning. The rotted part (at top) is full of starch grains. P'rom the sound part 
(below) the starch has been removed to form the cork-layer. C :)mpare with Fig. 
136. 




Fig. 175. — Cross-section of a potato stem inoculatefi with Bacilln.s carotovorus 
for comparison with Bacillus phi/tophthorus. Section far above the point inocu- 
late(i. \'essel full of bacteria. 



234 BACTERIAL DISEASES OF PLANTS 

in gelatin plates (X125) are irregularly spherical, often more 
or less clumpy, uniformly granular and with sharp margins 
which tend to become hazy. Sometimes the buried colonies 
send out colorless root-like growths (Fig. 182). Stab cultures 
liquefy first at the surf ace but eventually throughout (Fig. 183A). 
There is a fragile imperfect white pellicle and a copious white 
precipitate, the fluid becoming strongly alkaline. 

Peptonized beef bouillon clouds very rapidly, especially 
when neutral to phenolphthalein (in 6 hours at 30°C., when in- 
oculated with a 1-mm. loop). If undisturbed there is formed 
a very thin imperfect pellicle which shakes down readily leaving 
a . scanty interrupted rim, easily washed away. The white 






is 



- ■ \c ■■■ 

K' ■ ■ 

Fig. 176. — Flagellate rods of Bacillu>i carotovorus. From a 2-day agar streak. 
Van Ermengem's silver nitrate stain. In the upper right there are two bacterial 
rods lying together. X 1000. 

precipitate is not copious. It (3a) gives in +15 peptone 
bouillon a heavier clouding than B. phytophthorus. Growth 
in Dunham's solution is feeble. Growth in Uschinsky's solu- 
tion is abundant and long-continued and the fluid remains 
more or less acid throughout: there is a copious precipitate 
(15 or 20 times that in bouillon), but only a delicate easily 
fragmented pellicle. In milk a curd separates about the fourth 
day. It has the odor of cheese curds and there is little or no 
peptonization of this curd. Litmus milk is reddened and the 
litmus is, or may be, subsequently reduced. Other pigments 
are reduced, such as methylene blue (in Dunham's solution 
with grape sugar, not without). 



JONES' SOFT ROT OF CARROT, ETC.: CAUSE 235 



Fig. 177.— Agar poured plates of Bacillm carototwu.s? (Jones 3a?) on +15 
peptone beef agar at 25°C. for 10 days. Photographed Mav 3, 1919. Culture 
long in my laboratory and frequently transferred along with other soft rot isolations. 
Possibly confused in some transfer with Bacillus aroideae. This is the culture 
that was infectious to calla lily, and various other statements in the text respecting 
B. carotovorus are based on this organism.- 



236 



BACTERIAL DISEASES OF PLANTS 




Fig. 178. — Original stock of B. carotovorus (Jones' 3a, received from hi]u in 
1920). Photographed after 3 days at 25° on + 14 beef-pepton agar, showing 
surface and buried colonies. Y is a buried colony beginning to come to the sur- 
face, Z is a young thin colony. Photographed, X 10 by oblique transmitted light 
for comparison with Fig. 177. The colonies are smooth on the surface and show 
internal wavy markings liy oblique transmitted light. S'till infectious to carrot. 



JONES SOFT ROT OF CARROT, ETC.: CAUSE 



237 



The growth on steamed potato cylinders forms a slightly 
raised, wet-shining, smooth, cream-white covering, with a slight 
evolution of gas and the conversion of starch into amylodextrin. 





Fui. 179. — FtM'inentation tubes with potato juice: .4. Jones 3a (originiil 
Bacillun carotovorus) from Wisconsin in 1919. Gas in 24 hours. The gas in 48 
hours and in 15 days is also indicated. B. Jones 3a (long in my laboratory). 
No gas in 4 days. Amount of gas in 15 days is indicated. Good growth in both 
tubes. B rots calla lily, A does not. B also rots young carrot tops. 

Gas is also formed from steamed carrot cylinders and the gray- 
white bacterial layer is seldom thick enough to hide the orange 



238 BACTERIAL DISEASES OF PLANTS 

or yellow color of the substratum. Both these substrata become 
alkaline as early as the third day and increasingly so later on; 
both are softened, especially the carrot which often may be 
shaken apart in water after a week (Jones) or even in much less 
time. 

The maximum temper.^ture for growth is between 38° and 
39°C. The optunum temperature for growth is between 25° 
and 30°C. The minimum temperature for growth on steamed 
vegetables is above 4°C. Raw vegetables have not been re- 
ported upon.^ No appreciable growth was obtained on any 
medium at 0.6° to 1°C. (20 days) but there was a shght growth 
on nutrient gelatin at 2° and at 3°G. 

Growth after 5 days at 12°C. on steamed vegetables (potato, 
carrot, turnip, rutabaga) was about one-third that on the same 
substrata at 20° to 24°C. 

Except as already noted the cultures were free from strong 
odors. Not much indol is produced. 

Neutral bouillon gives the best growth, but the organism 
tolerates sodium hydroxid in bouillon down to below —40 on 
Fuller's scale and malic acid up to a little beyond +30. The 
organism is sensitive to it own acid products. In peptone 
water containing grape sugar, swollen, vacuolate and knobby 
involution-forms occur. 

Tolerates sodium chlorid up to 6+ per cent but not 7 
per cent in +15 peptone beef bouillon. Grows well in +15 
bouillon with 5 per cent NaCl. 

Jones 3a (culture received from him in 1919 — descend- 
ant of his original isolation of Bacillus carotovorus) tolerates 
ethyl alcohol up to 7 per cent in +15 peptone bouillon; grows 
promptly and well in the presence of 5 per cent. In further ex- 
periments it grew readily and formed a heavy pellicle in the 
presence of 10 per cent ethyl alcohol and made some growth 
in the presence of 11 per cent, but would not grow in the 
presence of 12 per cent. See Fig. 184 where the behavior 

1 In October, 1915, growth and rot were obtained by the writer on raw potato 
and carrot at 5°C. inoculating (3a) from a potato culture, but neither at 5°C. nor 
at 8°C., inoculating from bouillon. The first rot from the bouillon inoculations 
was at 9° to 11 °C. and that feeble (5 daj's). 



JOXES' SOFT ROT OF CARROT, ETC.: CAUSE 239 

of six soft-rot organisms is shown in +15 peptone beef bouillon 
containing ten per cent of Squibb 's absolute ethyl alcohol viz. 
3a recently from Jones (year 1919), 3a long in my laboratory 
(the branched 3a of Fig. 177), Potter's organism (Xo. 79 of 
Jones' laboratory), Spieckermann's organism (No. 78 of Jones' 
laboratory), Bacillus phytophthorus (Appel I) and Bacillus 
apiovorus Wormald. 





Fig. 180. Fig. 181. 

Fig. ISO. — Surface colony of Bacillus carotovorus on + 10 beef-peptone gelatin 
after 24 hours at 18°C., showing marginal fringe. X 110, about. Actual dia- 
meter of colony, 0.4 mm. 

Fig. 181. — Margin of a surface colony of BacilluH carotovorus after 3 days on 
gelatin, showing bacterial fringe pushing out into the gelatin. The darker band 
behind the fringe was liquefied and full of bacteria which exhibited, as a whole, 
a weaving or swaying motion. Medium magnification. 

XoTE. — Not having myself worked-over all of these soft-rot 
organisms critically, i.e., through a series of years, the following 
conclusions on the sjmonomy are expressed tentatively. 

Forms apparently identical with Bacillus carotovorus are 
Bacillus oleraceae Harrison on cauliflower and Bacillus omnivorus 
van Hall on iris. Under the name of Bacillus apiovorus 



240 



BACTERIAL DISEASES OF PLANTS 



Wormald in England has described a schizomyeete which at- 
tacks celery producing a soft-rot (Figs. 49 and 185) but is not 
active on potato shoots (Fig. 186). This, he is now inclined to 
think, is also identical with Bacillus carotovorus, but I am in 
doubt and shall keep it separate for the present. 

Very closely related forms are Bacillus aroideae Townsend 
(Fig. 187) on calla lily and Bacillus melonis Giddings on musk- 
melon. Townsend's organism differs in the form of its colonies 
on agar (they are, however, like those shown in Fig, 177) and 
in some of its fermenting powers, i.e., acid without gas from 
dextrose, lactose, saccharose and mannit. Its effect on raw 




Fig. 182. — Buried colonies of Bacillus carotovorus in +10 beef-peptone gelatin 
plates after 24 hours at 18°C., showing colorless root-like extensions. The several 
small dark spots ringed with light are due to irregularities in the gelatin or to 
dirt on the eyepiece. X 135 circa. 



carrot at the end of 8 days is shown on Fig. 188. Gidding's 
organism produces abundant gas from milk (99 per cent. COo) in 
the closed end of fermentation tubes; liquefies blood serum; 
does not produce gas with dextrose, saccharose, lactose, maltose 
or mannit, and has a maximum temperature of about 45°C. 

I think Bacillus aroideae and Bacillus inelonis are identical. 
At least an organism isolated by us from rotting calla lily and 
identified as Bacillus aroideae produces gas in milk (Fig. 189). 
With his original isolation (now lost) Dr. Townsend made no 
tests in fermentation tubes containing milk, 

I have been inclined to think that Bacillus carotovorus and 
Bacillus phytophthorus are not strictly identical, and have kept 



JONES SOFT ROT OF CARROT, ETC. : CAUSE 



241 



them separate in this volume, but further comparisons are 
necessary, 

Technic. — It is not difficult to isolate this organism, since 
very often it occurs almost unmixed in the decaying tissues. 
If the advancing margin of the rot is selected and the surface 
organisms are destroyed by pressing a hot spatula on the part 
selected (which ma}' be the sound surface near the rot), one may 
then dig through the burned surface and into the rotted area with 
little danger of external contamination and the certainty of ob- 




Fui. 183. — .4, Bacillua caroiovorua L. R. J., and B, Bacillus apivvoruH Worniald, 
in gelatin stabs at the end of 5 days at 20°C. 

taining on the poured plates almost or quite a pure culture of 
the parasite. The organism is easil}^ identified by its rapid 
disintegrating action on raw carrots or turnips and by its 
cultural peculiarities. As here described it can not be distin- 
guished with certainty from Bacillus phytophthorus (No. VII) 
by its behavior on. raw potato, nor by its growth in thin-sown 
gelatin plates, as I formerly supposed (compare Figs. 206 and 
212A with a, b of Fig. 205). 

For inoculation experiments, roots of various kinds may 
be selected and also fleshy above-ground parts. The work may 

16 



242 



BACTERIAL DISEASES OF PLANTS 




JONES' SOFT ROT OF CARROT, ETC. I TECHNIC 243 

be done either in the laboratory or in the hothouse. Owing to 
the rapidity of the rot, it lends itself very well to laboratory 
experiments, as the various susceptible organs can be inoculated 
and the results obtained before the plants begin to shrivel from 
exposure to unsuitable laboratory conditions. If whole roots are 
used, such as carrots, radishes and turnips, they may be exposed 
after inoculation either to the dry air of the room or may be 
put into deep uncovered jars. These latter serve better than the 
open air of the laboratory to hasten the earlier stages of the 
infection, but are not required to induce it. Full-grown or 
nearly full-grown roots are better for inoculation than immature 
ones. Such roots must not be flabby. 

As additional materials for inoculation, the student may 
use green cucumber fruits, which rot quickly, or green tomato 
fruits. He may also try the white, fleshy, central part of cab- 
bage stumps. 

By far the most convenient way, owing to the small space 
required, is to make the inoculations on thick slices of uncooked 
roots and fruits placed in deep, covered Petri dishes holding 
part of the slices in each dish uninoculated for comparison. 
For preparation of these slices which may be 2 to 3 centimeters 
thick, see Part II, page 103. 

Determine 

For THE ORGANISM. Morphology. — Size in microns 
(especially the diameter of the rods, most of which should 
measure 0.7 to 0.8/i). Absence of endospores (try heat and 
spore stains). Absence of capsules. Motility on the margin 
of a hanging drop (examine various cultures, old and young). 
Number and location of the flagella (Lowit's flagella stain, 
van Ermengem's stain). Occurrence of chains and filaments 
(examine second-day bouillon and agar cultures). Measure 
the longest filaments seen. Determine number of elements in 
the longest chains. Are the filaments and chains always, or 
usually, motile? Do pseudo-zoogloeae occur and, if so, in what 
media and under what conditions? Staining properties (using 
various basic anilin stains with and without mordants). Does 



244 



I5ACTERIAL DISEASES OF PLANTS 




Fig. 185. — A celery plant inoculated by needle-pricks with Wormald's Bacillus 
apiovorus in the central part. Inoculated May 11, 1915. Photographed May 
17. One inoculated leaf -stalk broken over, another diseased and discolored 
but still erect. 



JONES' SOFT ROT OF CARROT. ETC.: MORPHOLOGY 245 








Fig. 186.— Potato shoots inoculated 40 days at base with Bacillu.-^ apiovorus 
Wormald, showing slight effect. Inoculated by needle-pricks March 20, 1915. 
Photographed April .30. 



246 BACTERIAL DISEASES OF PLANTS 

the organism stain by Gram? Jones says it does, Harding 
and Morse say it does not. Examine always with the substage 
diaphragm wide open. For comment on use of Gram's stain 
see " Bacteria in Relation to Plant Diseases," Vol. I, page 188. 
Is it an acid- fast organism? Presence or absence of involution 
forms (examine 2 -months-old cultures on steamed carrot 
and read w^hat Jones says on page 314 in his first English paper). 
Try also much younger cultures in 1 per cent dextrose peptone 
water. Any Y-shaped or branching forms? Make cultures 
in bouillon containing 4 to 10 per cent of ethyl alcohol and 
search for the coccus-like forms which Wormald has described 
and figured for Bacillus apiovorus in "The Celery-rot bacillus." 
(The Jour. Agr. Science, Vol. VIII, Pt. II, March, 1917). 

Cultural Characters. — Determine behavior on thin-sown agar- 
poured plates, also in agar-streak cultures and stab cultures. 
In thin-sown gelatin-poured plates study (on the first, second 
and third days) the buried and the fimbriate-margined surface 
colonies with medium magnifications. Determine rate of 
liquefaction in gelatin stab cultures; behavior of streak cultures 
on Loeffler's solidified blood serum, on solidified egg albumen; 
curdling effects in milk and litmus milk; growth on steamed 
potato cylinders and carrot cylinders; behavior on the surface 
of raw potatoes in contrast to surface of raw carrots. Do you 
find any marked difference? Inoculated raw apple and raw 
turnip will afford another interesting comparison. If there 
is time, a whole series of raw fruits and vegetables (thick slices 
in deep Petri dishes) should be inoculated. Results cannot be 
compared, of course, unless the temperatures are the same. 

Growth in -fl5 peptone beef bouillon should be compared 
with that in salted peptone water (Dunham's solution), and 
in various synthetic media, e.g., Cohn's solution (no growth), 
Uschinsky's solution (abundant growth), Fermi's solution, etc. 

Contrast with B. phytophthorus in Soyka's milk-rice. The 
one culture (3a) should be yellowish, the other, pinkish.- 

Behavior in good fermentation tubes in peptone water 
containing various sugars and alcohols. With which is there 
clouding in the closed end? With which gas formation ? What 
is the composition of this gas? At least some simple experi- 



JONES' SOFT ROT OF CARROT, ETC. : CULTURAL CHARACTERS 247 




^ 



^ 




^ 



7^ 



■^ 



* 



Fk;. 1S7. — Flagella of Bacillus aroideae Townsend. Photographed by the 
writer March 29, 1915, from three fields on a slide stained by M. Katherine Bryan 
from a 24-hour agar culture using van Ermengem's silver nitrate method. Not 
Townsend's original isolation but one of our own from a diseased calla lily, proved 
up on caHa lily. X 1000. 



248 



15ACTERIAL DISEASES OF PLANTS 



ments can be tried, i.e., effect on the gas of shaking, in the presence 
of a strong solution of sodium hydroxid or potassium hydroxid. 
Is part of it absorbed? Is the absorbed part, roughly, one-fifth? 
Do not conclude too hastily. Allow time. Will the remainder 




Fig. 188. — A. Effect of Bacillus aroideae Townsend, on raw carrot, B. c!ieck 
one-half. Time, 8 days. The left part was stirred up a little with a glass rod. 
Inoculated in 191.5. 

explode when brought into contact with a flame? Is it hydro- 
gen? From what media are acids produced? Can the organism 
develop acid without gas (try glycerin)? Is more than one 



JONES' SOFT ROT OF CARROT, ETC. : CULTURAL CHARACTERS 249 

acid produced? Distill a flask-culture which has become acid 
and determine whether the steam is acid to neutral litmus paper. 
Use a large flask with a shallow layer of liquid. Collect the steam 
in water and make tests for nature of the acid. Is it acetic acid 
or only CO2? Boil the residue and determine whether it 
becomes more acid on concentration. Can you identify the 
residual acid? Is it lactic acid? 

Contrast with B. phytophthorus in +15 peptone bouillon 
with from 5 to 10 per cent of ethyl alcohol added (by means of a 
sterile pipette) after sterilization. 

Study nitrogen nutrition, reduction of nitrates, formation of 
hydrogen sulphide, ammonia, indol. Production of enzymes — 
starch-converting, proteolytic, cytolytic, etc. Whatispectinase? 
Try the following experiment: Inoculate the center (surface 
only) of several agar plates and when the growth has become ^i 
inch in diameter cut out the agar with a sterile knife in such a 
way as to remove all of the bacterial growth without touching it 
and transfer the agar bottom down to slices of raw carrots, tur- 
nips, etc. If you have done the work properly there will be no 
growth of the bacteria on the raw surface and yet it will rot. 
Why? Demonstrate absence of bacteria in the decaying tis- 
sues and describe their appearance under the microscope. 

Is the milk curd a normal cheese curd ? Is gas ever produced 
from milk? Try it in the closed end of fermentation tubes. 
Hold checks. 

On what raw media and steamed substrata is the brown pig- 
ment produced? What is the nature of this compound? Is it 
a host reaction or a bacterial excretion? 

It is important to isolate the organism from carrots (natu- 
rally rotting), from Gidding's melon rot and from Townsend's 
calla lily rot for comparison. Do so by all means if you have 
the opportunity, or send the material to some one who will. 
Much additional work remains to be done on the soft-rot 
bacteria. 

Non-nutritional Environment. — Action of heat, cold, dry air 
(very sensitive), sunlight vvery sensitive), acids, alkalies, germi- 
cides. Behavior in vacuo, and in neutral gases such as hydrogen, 
nitrogen, carbon dioxide. 



250 



BACTERIAL DISEASES OF PLANTS 




Fig. 189.— a. Fermentation-tube milk culture of Bacillus aroideae. The 
closed arm is full of gas (all CO2) and so is the U. From x upward, whey; and 
downward, curd. Curd also in base of the closed arm. Inoculated January 15, 
1915. Photographed February 10, 1915. 

B. Same as A but 48 hours later when all the gas has been absorbed by ad- 
dition of a strong solution of sodium hydroxide. 



SOFT ROT OF CARROT: NON-NUTRITIONAL ENVIRONMENT 251 

Why does 5 per cent grape-sugar retard or inhibit growth? 
Why does it ear y kill off the cultures grown in media containing 
it? ^ 

For the disease: Signs. — Write a description of the signs 
of this disease drawn from your own inoculations on carrots and 
other vegetables. How many hours from inoculation to the 
first appearance of the soft rot? Study the progress of the 
rot as related to: (1) copious vs. sparing inocu'ation; (2) moist 
vs. dry air; (3) cool vs. warm air; and (4) flabby vs. turgid tissues. 
At what cool temperature does the rot cease? Above what tem- 
perature does it cease? Compare this organism with No. IV, 
especially on raw potato, and with No. VII. 

Submit photographs or good drawings or both. Remember 
you cannot have too many striking and conclusive illustrations 
of the various diseases. 

Histology. — Fix, embed, section and stain early stages and 
late stages of the rot on various plants to determine location and 
action of the organism. Are the cells invaded or is the action 
of the organism entirely extra-cellular? Do not conclude too 
hastily. How are the middle lamellae destroyed, i.e., by tear- 
ing or by solvent action? What is the composition of this mid- 
dle part of the cell-wall? Is cellulose destroyed? How are 
the bacterial cavities formed? Are the cells killed in advance 
of the bacterial invasion ? Does the water-soaked area surround- 
ing a bacterial nidus stain the same in sections as the sound 
area just beyond (see Fig. 172 at .x)? Under the microscope 
unstained, does it look the same? What differences in the cell- 
wall? in the cell contents? In the attacked parts just previ- 
ous to disintegration are the cell-walls swollen? Make good 
permanent preparations showing various stages of the tissue- 
disintegration. 

Variability. — There appears to be considerable more resis- 
tance in some varieties of the attacked species than in others. 
Is this accidental or inherent? Why are green tomato fruits 
more subject than ripe ones? Why is the core of the carrot 
rotted sooner than the outer part? Why are young (seedling) 
carrots exempt? Are they, really? Why are the green parts 
of susceptible plants not more generally attacked? Read what 



252 BACTERIAL DISEASES OF PLANTS 

you can find on this subject and make some experiments. The 
man who is continually trying out his ideas by means of careful 
experiments is the one who makes discoveries. Reading 
alone will not serve; it makes a. full man, but not a. fruitful one. 
Transmission. — Nothing is known respecting special carriers 
of this disease. One should certainly avoid throwing diseased 
refuse into manure piles and into streams, and rotation of crops 
should be practised. Carrots should be dried and sunned as 
thoroughly as possible before storage, which should be at a low 
temperature. 

LITERATURE 

Read Jones: "Bacillus carotovorus n. sp., die Ursache einer 
weichen Faulniss der Mohre." Centralb. f. Bakt., 2 Abt., 
VII Bd., 1901, pp. 12 and 61; also Jones: "A soft rot of carrot 
and other vegetables, etc." 13th Report, Vermont Experiment 
Station, 1901; and Jones, and Harding and Morse: ''The bac- 
terial soft rots of certain vegetables" (23d Annual Report, 
Vermont Agricultural Experiment Station, 1910, where other 
literature is referred to.) 

Consult "Bacteria in Relation to Plant Diseases," Vol. I, 
Figs. 2, 3, and 88, and some statements in the text; also Ibid., Vol. 
II, text statements (see index). 

Read Spieckermann's " Beitrag zur Kenntnis der bakteriellen 
Wundfaulnis der Kulturpflanzen," Landw. Jahrb., 31 Bd., 
Berlin, 1902, p. 155, and Harrison's ''A bacterial rot of the 
potato caused by Bacillus solanisaprus'' Centralb. f. Bakt., 
2te. Abt., XVI, Bd., 1907, pp. 34, 120, 166, 384. 

See also Townsend's paper: "A Soft Rot of the CallaLily," 
U. S. Dept. Agr., Bureau of Plant Industry, Bulletin No. 60, 
47 pp., 9 pis., 7 text figures, 1904. 

And Gidding's paper: "A Bacterial Soft Rot of Muskmelon, 
Caused by Bacillus melonis n. sp.," Vermont Agricultural Ex- 
periment Station, Bui. No. 148, pp. 366-416, with 14 text 
figures, 1910. 

The first papers on this subject and the last one also are by 
Prof. L. R. Jones. 



VII. THE BACTERIAL BLACK ROT OF THE POTATO 

(Syn. Black leg, Basal Stem-rot and Tuber-rot) 

Type. — This is a wide-spread and very destructive soft rot 
of the potato and some other plants. Vessels are sometimes 
occupied, but it is a disease of the parenchj^ma rather than of 




Fig. 190. — Curling of potato leaflets due to Bacillus phytophthoru.s (.\ppel I). 
Spring of 1915. Second set of needle-prick stem inoculations. 

the vascular system, being found, according to Dr. Appel, only 

occasionally and exceptionally in the vessels of the potato plant. 

The first above-ground signs of the disease are either sudden 



254 



BACTERIAL DISEASES OF PLANTS 



wilting or a slow yellowing of the lower leaves and a stricter 
habit of growth in the upper ones, the leaflets of which (Fig. 190) 
are or may be more or less incurled (upward). If one examines 
the base of such shoots they will be found to be black-spotted 




f -v 



^ 



Fig. 191. Fig. 192. 

Fig. 191. — Stems of Green Mountain potato inoculated 48 hours at A' with 
Bacillus phytophthorus Appel, from a 2-day agar-streak culture. Stems black and 
rotting in the pricked area. Hothouse experiment of January 23, 1915. Tubers 
half grown. Organism from Germany. In the laboratory since August, 1906. 

Fig. 192. — Same lot of plants as Fig. 191, but 4 days after inoculation (at A'). 
Stems black and nearly rotted off in the pricked area; bundles infected upward 
for long distances and with a brown stain coming to the surface. 

end more or less softened (Figs. 191, 192) at the surface of the 
earth or just below it, and hence the German name Schicarzbein- 
igkeit (black leg). Generally at first this blackening and ulcera- 
tion are restricted to the base of the stem but upper parts soon 



BLACK ROT OF THE POTATO: TYPE 



255 



hH 



wilt, blacken, shrivel, and fall over 
(Fig. 193), and often the tubers decay. 
The disease is readily inoculable into 
the soft upper part of shoots (Fig. 194) • 
and may run out on the petioles in 
black lines exactly as in case of Bacter- 
ium solanacearum (No. IV) or of Bacil- 
lus amylovorus (No. XII). It is also 
readily inoculable into the base of 
potato shoots when not too old (Figs. 
195, 196) and then progresses in the 
same way as the natural infections. 
Old shoots are less susceptible than 
young ones (Figs. 197, base, and 198) 
and very often when the basal parts 
of stems are inoculated they rot across 
and break over without much down- 
ward movement of the bacteria (Fig. 
198), the underground parts sending 
up new dwarfed shoots to take the 
place of those destroyed (Fig. 199). 
Prior to shriveling, especially as the 
season advances, all the green parts 
of the potato (stems, leaves, flower 
stalks) may show black spots due 
to the organism and from these 
pure cultures of it may often be ob- 
tained (Appel). The bacteria neither 
show any special tendency to multiply 
in the vascular bundles, although some- 
times found there (Fig. 200), nor, in fig. 193.— Potato plant 

inoculated by needle pricks 

on the base of the shoot (at X) and wilted by Bacillus phytophthorus (Appel I). 
Plant inoculated January .30. 1915 from a 16-day culture on a steamed potato. 
Photographed on the 7th day, 1-5 natural size. Of 9 shoots on 6 plants all of 
which were inoculated only one failed to become diseased, and this was one of 4 
from the same tuber (perhaps it was older than the other 3 shoots). The disease 
progressed much faster upward than downward as shown by the tiny shoot which 
is still unaffected though coming out of the base of the blackened stem at the 
earth's surface. Earlier stages of the disease resembled Figs. 191 and 192. 



256 



BACTERIAL DISEASES OF PLANTS 




Fig. 194.— Potato shoot from same lot as Fig. 193 {Bacillus phijtophthorus) 
but from another plant and inoculated at the top. Hothouse temperature 
ranging from 65°F., to 95°F. Needle-pricks at X, from a 2-day agar-streak 
culture. Time, 43 hours. German organism (Appel I). 



BLACK ROT OF THE POTATO: TYPE 257 

my top-inoculated plants have I seen any strong tendency of 
the disease to extend into the tubers such as we see in case of 
potato shoots inoculated with Bacterium solanacearum. The 
disease occurs in its worst form during warm moist summers and 
autumns, but may continue on the tubers through the winter, if 
the temperature in the store-houses or pits is sufficiently high. 
In the tubers, the disease does not begin in the vascular ring 
(contrast with No. IV). In the laboratory raw potato tubers 
may be rotted very quickly (contrast again with No. IV) by 
streaking the organism on their cut surface (Fig. 201). Lenticel 
infections occur on the tubers (Fig. 202). Starch is not de- 
stroyed and the infection is chiefly intercellular (Fig, 203). 

Dr. Appel's principal studies were made upon the potato 
but he also isolated his organism from diseased comfrey {Sym- 
phytum officinale). He successfully inoculated it into yellow 
lupins, horse beans {Vicia faba), green tomato fruits, slices of 
raw carrot, etc. 

This disease occurs all over Germany, in Ireland, and in 
various parts of the United States (Maine, Virginia, South 
Carolina, Wisconsin). Its distribution is probably co-extensive 
with the culture of the potato, and I now regard it as one of the 
most serious diseases of the potato. 

Cause. — The basal stem-rot is due to Bacillus phytophthorus 
Appel (not sufficiently distinguished from Bacillus carotovorus 
Jones, which name is earlier). This is a white, rapid-growing, 
non-sporiferous. Gram negative, motile, peritrichiate-fiagellate 
(Fig. 204), promptly liquefying, nitrate-reducing, aerobic and 
facultative anaerobic, acid-forming, gas-forming (but not in 
the potato), milk-curdling (by formation of an acid), alkali- 
tolerant, sodium chlorid-tolerant, chloroform-tolerant, dry- 
air-sensitive, rod-shaped or filamentous schizomycete, forming 
quickly on agar-poured plates circular, grayish white or, by 
transmitted light, slightly bluish white, well-developed colonies; 
and on very thin-sown gelatin plates characteristic, rapid-grow- 
ing, big, circular (Figs. 205 and 206), opaque- white, fringed 
(fimbriate-margined) colonies (Fig. 207), floating in a pit of 
liquefaction (thick-sown plates liquefy too rapidly for these 
examinations). The buried colonies in gelatin plates appear as 

17 



258 



BACTERIAL DISEASES OF PLANTS 




BLACK ROT OF THE POTATO: CAUSE 



259 




u 






b[) a 



oq 



p^ 



260 BACTERIAL DISEASES OF PLANTS 

shown in Figs. 208 and 209. The organism is white on most 
media but on Soyka's milk rice it is pale pinkish cinnamon 
verging toward vinaceous pink in old cultures (R2). Streaked 
on slices of raw potato it grows rapidly and characteristically, 
forming a white slime surrounded by a dark (black or brown) 
border in the disintegrating flesh of the potato. The disin- 
tegrating potato shoots also are often very black. Bouillon is 



1/ 



;\ 




Fig. 197. — Base of Fig. 193, 2 days later. The mother tuber, the young tubers 
and the woody base of the stem are still sound. 

clouded very quickly and gelatin stabs develop a prompt 
funnel of liquefaction. Potato juice clouds quickly even in 
the absence of air (closed end of fermentation tubes) but no 
gas is formed. Ethyl alcohol in peptone bouillon retards 
or hinders growth (Fig. 210A). The organism does not form in- 
dol, and does not grow in Cohn's solution. It produces a non- 
volatile acid from dextrose, saccharose, lactose, galactose, and 



BLACK ROT OF THE POTATO: CAUSE 



261 




— 02 



— 2 
i >> 



it [^ 



:^ ^ .„• 






_£ -5 (N 



262 



BACTERIAL DISEASES OF PLANTS 




BLACK ROT OF THE POTATO: CAUSE 263 

maltose, and small quantities of gas from innosit (muscle sugar), 
lactose and mannit. A volatile acid also distills off from pep- 
tone dextrose cultures (flasks 15 days old). It is a common be- 
lief (of German origin) that the organism.loses virulence readily, 
but in nine years, in the strain originally received by me from 
Dr. Rudolph Aderhold in Berlin, and designated in my labora- 
tory as ''Appel I," I have not observed any loss of virulence. 
The last inoculations made with it by me in June, 1915, on 
rapidly growing potato shoots by needle pricks, yielded striking 




Fig. 200. — Cross-section of potato stem several inches above the inoculated base. 
Enlarged to show the bacteria {Bacillus phytophthorus) occupying a vessel. 

infections, the tops of the 18 inoculated shoots being entirely 
destroyed in 5 to 7 days.^ A contaminating non-parasitic coccus 
form is common (Appel). This is, probably, what Dr. A. B. 
Frank figured, and supposed to be the parasite. 

Technic. — Because saprophytes quickly follow parasites in 
the decaying potato, it is often difficult to isolate the latter, 
Bacillus phytophthorus being no exception. This sufficiently 
explains why the causes of such an insistent and annually re- 
current phenomenon as the rot of potato tubers remained so long 
undetermined. Not knowing that the same organism could not 
both begin and complete the destruction of the potato tuber, 

1 1 tested it again on potato tops in April, 1919 (13th year in my laboratory) 
with the same striking results. See Fig. 211. It was also infectious in 1920. 



264 BACTERIAL DISEASES OF PLANTS 

Reinke and Berthold, Kramer, Frank, Wehmer, and, prior to 
Appel, all the Germans who studied potato rots (mostly by 
means of the microscope) were led astray b}^ the non-infectious 
saprophytes (starch-destroyers, gas-producers, endospore-bear- 
ing rods, coccus forms, etc.) which soon swarm in the tissues 
and complete their destruction, but cannot be induced to begiii 
the rot except under very abnormal asphyxiating conditions. 
Then, of course, almost any refuse-loving organism will grow 
in the dead tissues, since the potato is a good culture medium 
for many things. In this connection, read Wehmer 's paper in 




-.'^r^ 



Fig. 201. — Streak culture of Bacillus phyiuplithorus Appel on raw potato 
36 hours at about 23°C., showing a soft rot bordered by a dark stain. Inoculated 
from a 48-hour culture on steamed potato. Tuber soaked 30 minutes in 1:1000 
mercuric chlorid water before cutting with a sterile knife. The check }2 remained 
sound. Laboratory experiment of 1914. i'2 nat. size. 

Centralblatt f. Bakteriologie, 2te Abt., IV Bd., 1898, pp. 540' 
570, 627, 694, 734, 764 and point out his fallacies. 

Appel by his masterly paper (1903) let a flood of light 
into an obscure situation, because while' the writer had proved 
conclusively 7 years earlier (1896) that a part of the potato rot 
of the United States was due to a schizomycete, this organism 
(Bacterium solanacearum) had not been subsequently isolated 



BLACK ROT OF THE POTATO: TECHNIC 



265 



with certainty from European potatoes, and its existence^did 
not account for all of our own potato rots, and particularly for 
those common in the more northern parts of the United States 
in which the distribution of Bacterium solancearum remained 
(and still remains) unknown, although it was isolated by us 
nearly every year from various species of plants received from 
our Southern States. 




Fig. 202. — Potato tuber showing lenticel infections due to Bacillus melan- 
ogenes. At the bottom on the left side and in the center, various infections have 
fused. From a plunge inoculation by the writer in 1912. 

. Following the appearance of Appel's paper, his organism 
was found in the United States by the writer and others (Morse, 
Jones, etc.), and two or three similar organisms were soon 
described, e.g., Bacillus solanisa'prus Harrison. Morse has 
substituted van Hall's name, Bacillus atrosepticus, for Appel's 
name but a careful re-reading of van Hall's Dutch paper (May 



266 



BACTERIAL DISEASES OF PLANTS 



21, 1902) leaves me in doubt. No one now has transfers from 
van Hall's original culture, I believe, so as to enable one to clear 
up the doubtful points and under the circumstances it is best, 
I think, to retain Appel's name, especially as van Hall made 
very few inoculations under natural conditions and as he says 
of his organism: ''On artificial media the parasite loses its 
virulence very quickly." Moreover, if May 21. 1902, or some 







Fig. 203. — Stained section of a potato tuber in the vicinity of an infected 
lenticel (stage of Fig. 202) showing bacteria dissolving the middle lamella and 
wedging apart the starch-bearing cells. 

later date is the actual date of publication of van Hall's thesis, 
then Appel's name is at least 2 months earlier than van Hall's 
name and the latter, even if synonymous, does not have priority. 
To isolate this organism, the surface through which it is 
proposed to enter for cultures should be burned with a hot 
knife or spatula, or soaked for twenty minutes in 1 :1000 mercuric 



BLACK ROT OF THE POTATO I TECHNIC 267 

chlorid water. Bj^ preference the entrance should be through 
sound tissues close to the advancing margin of the rot, from 
which scrapings may then be made for the poured plates. 
One should not be discouraged if the first plates yield only 
saprophytes, but should try in several other places on the same 
plant or on other plants. 

The organism grows readily and is easily identified by its 
behavior in Cohn's solution, litmus milk, thin-sown gelatin 
plates, surface of raw potato, etc. Spore-bearing organisms 
and coccus forms may be eliminated from consideration on the 
start, also those schizomycetes that produce gas from potato 



• 




:1 

■ r 


■^ 




. V ' _ 


4 







Fig. 20-4. — Flagellate rods of Bacillus plujtophthorus Appel. Stained from a 
young (2 day) agar culture of Appel I by van Ermengem's silver nitrate method. 
Photographed March 29, 1915. X 1000. (Compare with Fig. 176.) 

juice in the closed arm of fermentation tubes, and all vile-smelling 
forms. 

For inoculation it is best to select the base of young shoots of 
the potato in rapid growth, or the soft tops of older plants, using 
needle-pricks from young cultures on agar, potato or gelatin. 

Tubers to be inoculated should be freshly dug (or at least 
not flabby), sound and flawless, and may be kept either in the 
open air of the laboratory or in damp air under bell-jars. If 
many checks are held it is sufficient to wash the surface free from 
dirt. If none are held, then the part through which the needle 



268 BACTERIAL DISEASES OF PLANTS 

enters must be soaked in 1 :1000 mercuric chlorid water for 40 
minutes. 

In wet autumns, in moist soil, there is often a wholesale 
rotting of potato tubers due to the entrance of the parasite 
through the lenticels and it is a very instructive experiment to 
demonstrate lenticellate infection in the laboratory. Sorauer 
was, perhaps, the first person to see bacteria enter the potato 
tuber in this manner. The writer saw it many years ago 
(1886) and has obtained on potato tubers in recent years very 
typical and beautiful infections by way of the lenticels (Fig. 202), 
using Bacillus melanogenes (which as received by me was a mix- 
ture of Bacillus phytophthorus and Bacillus solanisaprus) . 
For this purpose one should select smooth, sound, recently 
harvested tubers, wash clean and plunge for 30 hours under 
distilled or autoclaved or even non-sterile tap water, to which a 
young agar-streak culture of this organism has been added. 
If the variety is susceptible, numerous infections centering in 
lenticels should appear within a few days, and soon the interior 
of the tubers should be wholly disintegrated. The sterility of 
the water used is of no great consequence so long as it does 
not contain parasites, and so long as the surface of the tuber 
itself is not sterile, the aim being to set up conditions like those 
obtaining in ordinary infections in wet earth. Of course, checks 
to w^hich the parasite has not been added should be held and 
these will remain sound unless their surface happens to be 
contaminated with this or some similar parasite, but the tubers 
must not be asphyxiated. 

What proportion of the wholesale rot of potato tubers in the 
soil in wet autumns is to be ascribed to Bacillus phytophthorus, 
BqcHIus solanisaprus, and similar bacterial parasites, and what to 
mere saprophytes following asphyxiation cannot be determined 
until a great many more studies have been made of the flora of 
rotting potatoes. 

Determine 

For the organism: 1. Morphology. — Size in microns, 
form, aggregation of elements (Do chains or filaments occur? 
See No. VI), motility on margin of a hanging drop (How long 



BLACK ROT OF THE POTATO: MORPHOLOGY 



269 



does motility persist in cultures?), presence and distribution of 
flagellte (use Hugh Williams' stain), absence of endospores (try 
heating for 10 minutes at 80°C., and spore stains). Any 
capsule? Reaction to Gram's stain (examine with the dia- 
phragm wide open). Is the organism acid-fast ? Do involution 
forms occur? Test in dextrose peptone water. 

2. Cultural Characters. — Growth on very thin-sown -j-lO 
beef-peptone gelatin plates at 20°C. is very characteristic 
(If the plate contains only 4 to 6 bacteria, they should grow 
quickly, forming big circular opaque white colonies (Fig. 20G) in 
3 or 4 days. Compare with No. \T (Fig. 212^.) The rate of 




Fici. 205. — a. 48-hour gelatin colonies of Bacillus carotoi'urus L. R. Jones, 
grown at same temperature and on same batch of gelatin as b. Natural size. 

b. Gelatin colonies of Bncillun phytophthorus Appel. Photographed natural 
size after 48 hours at 18°C. 



liquefaction in gelatin stabs resembles that oi Bacillus carotovorus. 
Appearance in agar plates, streaks and stabs. Growth on 
steamed potato. Behavior on slices of raw potato (select 
smooth, sound tubers, wash, soak 40 minutes in 1 :1000 mercuric 
chlorid water, slice when dry with a sterile knife, and put into 
shallow-covered culture dishes or deep Petri dishes) . Why does 
the black stain not appear also in the cultures on the cooked 
potato? What is tyrosinase? Does it play any part here? 
Why does the dark stain disappear in later stages of the rot in 
raw tubers (Fig. 201)? Growth in bouillon, nitrate bouillon, 
Cohn's solution, Uschinsk3^'s solution, Fermi's solution (Does 
this medium become yellowish or greenish?). Growth in milk 
(Is the casein thrown out of solution by an acid or by a lab 
ferment? What is the odor of the fermented milk? Does this 



270 



BACTERIAL DISEASES OF PLANTS 




Fig. 206.— Thin-sown +10 beef -peptone gelatin plates of Bacillus phy- 
tophthorus isolated from a South Carolina potato in 1917. Colonies exactly like 
"Appel I" from Germany, fi natural size. 



BLACK ROT OF THE POTATO I CULTURAL CHARACTERS 271 

organism produce a bad odor on any medium?); What is the 
early and late behavior of this organism in lavender-colored 
litmus milk (watch closely from the start) ? On a proper use of 




Fig. 207. — Bacillus phyiophthorus (Appel Ij. On +10 beef-peptone gelatin 
held for 3 daj^s at 16°C. All but the corona has liquefied. Actual diameter 
of colony, 1 mm. Margin fringed like Bacillus carotovorus (see Figs. 180, 181). 
Photographed February 4, 1915. Buried colonj^ below at left. The specks 
scattered about are due to dust on the eyepiece. 

litmus in milk, consult "Bacteria in Relation to Plant Diseases," 
Vol. I, pp. 48, 196. Test shake-cultures in beef-peptone agar 
(for gas-bubbles which will appear only if muscle sugar is 





Fig. 208. — Bacillus phywphthorus (Appel I). Small buried colonies from 
same gelatin plate as Fig. 207 but enlarged about one-third more. One colony 
out of focus. 



present) ; try peptone water in fermentation tubes with all the 
common sugars and alcohols. From which is acid only pro- 
duced? From which both acid and gas? 



272 



BACTERIAL DISEASES OF PLANTS 



If fermentation tubes are not available, shake-cultures 
may be made in litmus-peptone agar containing 5 per cent of 
the carbon compound to be tested (examine early and frequently 
for gas-bubbles and change of color). Study behavior in potato 
juice in fermentation tubes. The closed end should cloud. Is 
any gas formed? I have never seen any. Test suitable cul- 
tures for indol, for hydrogen sulphide. What is the nature of the 
acid, or acids, formed by this organism? Can they be driven 
off by boiling (test the vapor with neutral litmus paper and ob- 




FiG. 209. — Small buried ooloiiy uf BacUlm phytophthorus (Appel I) after 
5 days in gelatin at 16°C. The fringe looked like individual bacteria but con- 
sisted of lenticulate colonies as determined by staining in situ. Colony about 0.4 
mm. in diameter. Not all the buried colonies showed this fringe. 

serve the litmus reaction of the concentrated fluid) ? Is lactic 
acid produced? Determine toleration of acids. The organism 
is sensitive to acids (Appel). 

3. N^ on-nutritional Environment. — Effect of heat? of dry air? 
of sunlight? of freezing (salt and pounded ice)? of salted bouil- 
lons (try 5 per cent first)? of chloroform in bouillon? of weak 
acids? of sodium hydrate (try — 40 first)? of germicides? 

Can you get any growth on culture-media at 5°C. or at 
40°C.? Try several sorts, e.g., potato broth, peptone-beef 



BLACK ROT OF POTATO: NON-NUTRITIONAL ENVIRONMENT 273 




Fig. 210.— a. Bacillm phytophtlwrus in +15 peptone-beef bouillon with 
varying per cents of ethyl alcohol (4 to 7 per cent). Clouded check tube at the 
right, time 6 days. The others clear. B. Bacillus carotovorus (old 3a) ditto. 
All showing growth. The white at the bottom of the tubes is a reflection, not a 
precipitate. C. Same as B, but photographed against a white background. 
Introduced to show how the camera may deceive. 

18 



274 BACTERIAL DISEASES OF PLANTS 

bouillon, milk, steamed potato, nutrient agar, etc. Inoculate 
by needle punctures susceptible tubers (of more than one vari- 
ety) and place one or more of each sort at the following tempera- 
tures: 5°C., 8°C., 20°C., 30°C. Repeat if you are not satisfied. 
What do you conclude? Is there any practical application? 
If you have time, try also dry storage vs. damp storage at differ- 
ent temperatures, inoculating as before. 

Select tubers of some variety whose flesh reddens or browns 
quickly on exposure to the air, pare, grate quickly, squeeze the 
juice at once through a cheese cloth into a narrow, tall jar, 
divide into two equal portions, and steam one immediately 
(to destroy the action of the oxidizing enzyme) ; allow the other 
portion to oxidize freely in a shallow dish for 6 hours or more, 
then steam. The two lots may now be tubed, re-sterilized and 
comparative inoculations made, using, for each : a, one carefully 
measured 1-mm. loop from a very young fluid culture; h, the least 
quantity that can be withdrawn by dipping the end {lio centi- 
meter) of a platinum needle into the culture fluid. Watch the 
early stages of growth critically. What do you conclude? 

For the disease : (1) Signs. — What is the period of incuba- 
tion — ^on stems of different ages? on tubers? Time between 
local rot on the base of the inoculated shoots and a general ap- 
pearance of disease? What changes occur in the foliage? How 
soon after the above-ground signs do the tubers begin to rot? 
This is best studied in the open field in summer and autumn. 
Can the tops be destroyed without causing a rot of the tubers? 
How do you account for this ? Is the brown or black stain ever ab- 
sent from the stems when this organism is present ? There is a 
bacterial rot in which the brown stain is absent (Appel). To 
what is this colorless rot due? Describe the disease. What 
are the signs on the tomato? Try inoculating green tomato 
fruits. Is it a common disease of the latter? Can other plants 
be infected? Try all the plants that are rotted and not rotted 
by Bacillus carotovorus. Study the flora of many naturally 
rotting potatoes, especially the advancing margin of the rot, 
for the presence of this organism. 

Histology. — Section diseased stems and tubers and make per- 
manent mounts. Do the bacteria follow the vessels, or only 



BLACK ROT OF POTATO: HISTOLOGY 



275 




276 



15ACTERIAL DISEASES OF PLANTS 




Fig. 212.— a. Thin-sown ( + 10) peptone-gelatin poured plate of Bacillus 
carotovorus. Kept for 4 days at 1S°C. Compare with Figs. 205, 206 of Bacillus 
phytophlhoras. 



BLACK ROT OF POTATO! HISTOLOGY 277 

occupy them incidentally to the general destruction of the paren- 
chyma? Compare with sections of potato plants attacked by 
Bacterium solanacearum. Study the disintegration of the tuber. 
Are cavities formed? What is the earliest stage of the rot? 
What becomes of the starch? Of the cell-wall? Is any gas 
formed out of the tissues of the potato? Compare with Bacillus 
carotovorus (No. VI). Inoculated, rotting tubers placed at 5°C. 
for 10 daj^s offer a good opportunity to study the reduction of 
bacterial growth and the development of a protective cork 
barrier (See also No. VI). Cut sections until you have made 
out the newly formed cork layer clearly. Use some good cork 
stain if necessary. Make sections through lenticels on the tuber 
in very early stages of infection to show the bacterial penetra- 
tion. Cut on the microtome from paraffin-infiltrated material. 
These sections should be made not much later than the third 
day, i.e., as soon as a trace of infection is visible on the surface 
(using a hand lens). A few days later the lenticel infection is 
much more conspicuous, but then the bacteria have generally 
passed several millimeters beyond the region of the lenticel. 
Determine if you can how the bacteria enter the stem, i.e., are 
the stem infections stomatal, or only by way of wounds? Spray 
and look for stomatal leaf- infections. Appel left this matter 
undetermined. I failed in two attempts. 

Variability.— AW varieties of potatoes are said to be subject 
to this disease. Have you been able to find differences, either 
from your inoculations, or from field observations? Consider- 
able time devoted to such an inquiry might be well spent. It 
certainly would be if practical results were forthcoming, or even 
suggestions toward such results. The Early Rose, Imperator, 
Maercker, Magnum Bonum, and Wohltmann (sorts commonly 
grown in Germany) are frequently attacked (Appel). The 
writer found Daisy, Green Mountain, and Factor quite suscep- 
cible (see "Bacteria in Relation to Plant [Disease," Vol. II, 
Plate 1 1 — facing p. 96). White McCormick is also quite suscep- 
tible (Figs. 195 and 211). Are potatoes the fleshy part of which 



B. Cross-section of stem of Tropaeolum majus (nasturtium) attacked by 
Bacterium solanactarum, showing brown stain and bacterial ooze. Plant from 
Baltimore, Md. Photographed July 22, 1914. X 8 circa. 



278 ]}ACTERIAL DISEASES OF PLANTS 

darkens rapidly on exposure to air more resistant to this disease 
than those which possess this property to a feeble degree? Are 
those varieties whose lenticels open freely in wet soil specially 
subject to this rot? I believe they are. 

Transmission. — In two localities Appel saw this disease 
develop severely in places where potato refuse from rotten pits 
had been thrown out (for reference to a similar observation on 
black rot of the cabbage see No. II) and in one of these places 
the soil was still infectious after 5 years (Appel I.e., p. 387). 
Appel also found that sound-looking tubers from diseased fields 
often carried the infectious organism on their surface. Should 
rotting potatoes be left in the field? Should they be thrown on 
the dung heap, or fed to stock? What do you conclude respect- 
ing selection and treatment of seed tubers? What respecting 
early digging of the tubers, i.e., before fall rains have set in? 
What respecting the necessity for dry and eool storage, especially 
in years when the rot is very prevalent? 

The organism is sensitive to dry air and for this reason pota- 
toes should be dry when stored to avoid further rot in pits and 
cellars. 

Mr. Melhus states that he has combated this disease in the 
field very successfully by persistently pulling out the diseased 
hills and exposing them to the light and air (oral communi- 
cation). 

My own experiments lead me to believe that well-drained 
fields should be much less liable to attacks of the tuber rot in 
autumn than those which become water-logged following heavy 
rains. 

Rot due to Baeillus phytophthorus ceases at 4°C. and below 
8°C. (46°F.) it is slow. Potato tubers from fields where this rot 
has prevailed should therefore be stored at low temperatures 
and disposed of early. 

LITERATURE 

Consult Appel's paper ''Untersuchungen uber Schwarzbein- 
igkeit und die durch Bakterien hervorgerufene Knollenfaule der 
Kartoffel" in Arbeiten aus der Biol. Abth. f. Land.-u. Forst- 
wirthschaft am Kaiserl. Gesundheitsamte, Band III, Heft 4, 



BLACK ROT OF POTATO : LITERATURE 279 

Berlin, 1903, pp. 364-432. His first paper on this disease is 
in Ber. d. d. Bot. Ges. XX Bd., Heft 1, 1902. 

Read the writer's account of this organism entitled "Bacillus 
phytophthorus Appel," in Science, N. S., Vol. XXXI, May 13, 1910, 
pp. 748-751; Morse's paper in Journal of Agricultural Research, 
Jan. 15, 1917, p. 79; also Morse's earlier paper. Bull. No. 174, 
Maine Agr. Exp. Sta., Dec. 1909; and Rosenbaum and Ramsey's 
paper on "Influence of Temperature and Precipitation on the 
Blackleg of Potato," Journal of Agricultural Research, Vol. 
XIII, No. 10, Washington, D. C, June 3, 1918, pp. 507-513. 

For reference to the paper by Pethybridge and Murphy see 
the foot note on page 64. 

The name Bacillus phytophthorus was first published by 
Dr. Appel in 1902, in Ber. d. d. Bot. Ges., XX Band, Heft. 2, 
pp. 128-129. Note read Feb . 28 and Heft published March 27. 



VIII. THE BEAN BLIGHT 

(Syn. The bacterial bean spot) 

Type. — This is a disease of beans common on leaves, stems 
and pods, and confined principally to the parenchyma although 
the vessels also are invaded, sometimes for a distance of several 
inches. It occurs on several species of beans (Phaseolus) and 
is a serious disease. Whether other related genera are subject 
remains uncertain. Similar looking bacterial diseases occur 




Fig. 213. — Portion of under surface of an immature bean leaflet showing 
stomatal infections (light spots) due to a pure culture spray inoculation 
of Bacterium phaseoli isolated from an Idaho bean. Time, 3 days. Spots trans- 
lucent but not yet brown or sunken. Planar enlargement by James F. Brewer, 
September 26, 1914. X 8. 

on cowpea (Vigna) and on soy bean (Mucuna), but my cross- 
inoculations to plants of these genera failed (one trial only, 
but using many plants and virulent cultures sprayed on the 
foliage). A yellow organism resembling this one on agar and 
potato was plated from spots on leaves of the soy bean in my 
laboratory in 1902 from Charleston, South Carolina, and Wash- 
ington, D. C, and again in 1917 from Norfolk, Virginia. 

280 



V.-. 



THE BEAN BLIGHT: TYPE 



281 




Fig. 2\^.— Bacterium phaseoii: A pure culture, spray inoculation on a bean 
leaflet. Time, 3 days. Done by the writer in 1914. X 2. 



282 BACTERIAL DISEASES OF PLANTS 

The disease is first visible on the leaves a few days after 
infection (Fig. 213) is the form of minute translucent dots 
which gradually enlarge (Fig. 214) and from being slightly protu- 
berant become sunken and discolored, forming irregular reddish, 
yellowish or brownish spots (Fig. 215). Frequently on the yellow- 
ing leaves the green of the leaf persists around the spots (Fig. 216). 
As in cotton leaves attacked by Bacterium malvacearum, there 
may be distortions of the leaves due to disease of the veins (Fig. 
217). This occurs, so far as I have observed, only when infection 
takes place very early, i.e., when the leaflets are quite small. 
When older leajes are sprayed with a sus- 
pension of this parasite they become badly 
spotted but are not then distorted. 

On the pods the spots appear the sixth 

to eighth day as small (0.2 mm.) circular 

areas centering in a single stoma and are 

deeper green than the surrounding tissue 

(Figs. 218 and 219). These spots enlarge 

slowly (Fig. 220) being level or slightly 

y^ protuberant at first, as on the foliage, then 

W . > sunken and discolored and showing some- 

» times a reddish border. As the center of 

J. oi - T3 the spot shrinks (over the internal cavity), 

tiG.21o. — Bean ^ '^^' 

leaflet attacked by Bac- from destruction of the subjacent tissues, 
terium phaseoli. From bacteria from this cavity (as in the black 

a garden in Wasliington, ^ ^^ ^f ^j^^ j^^^^^^ ^^.^ {^^^^^ through the 
D. C, June, 1908. One- i i i • n i i 

half natural size. stomata abundantly, especially when the 

spots are on the pods (Figs. 221 and 222). 
These extrusions appear in the form of yellowish cirri, if the 
surface is dry, and of expanded masses or crusts, if the surface is 
alternately wet and dr^^ 

The bacterial multiplication in the leaves being less abundant 
than in the pods there is less surface ooze, but almost always 
there is some. Even in early stages of the leaf-spot, the bac- 
teria in the tissues are veiy abundant as shown in Figs. 223, 
224. The leaf-spots which are circular at first frequently coalesce 
as they enlarge, forming irregular areas which are often of con- 




THE BEAN BLIGHT: TYPE 



283 



siderable size and which usually retain a water-soaked trans- 
lucent) border. 

In severe cases the leaves shrivel and fall off, and the pods 
become worthless — spotted or dwarfed. If the weather is damp 
both leaves and pods may also become moldy. If attacked in 







Fig. 216. — First series of inoculations from the Idaho bean (September 23, 
191-ij, showing numerous stomatal leaf -spots due to Bacterium phaseoli. The 
leaf was yellow except around the spots, where the leaf-green persisted. Time, 
13 days. Temperature of hothouse, 65'C. to 95°C. Leaflet about one-third 
grown when sprayed. Xat. size. 



early stages of growth the leaves become curved, twisted and 
variously distorted by reason of injury to the developing veins 
(see Nos. IX and XI for similar phenomena) . The attacked leaves 
also exhibit a curious persistence of the leaf green around the 
spots while it disappears altogether from the rest of the leaf. 



284 15ACTERIAL DISEASES OF PLANTS 

On the fruit, the fleshy portion of the pericarp is the chief 
seat of the disease (Figs. 225and226),but the bacteria also burrow 
inward and often infect the interior of the pod and the surface 
of the ripening seeds. The seed itself may also be attacked. 

When the infections are through stomata, the first signs 
of the disease on the pods are minute, green spots, each surround- 
ing a stoma. Although very small in this stage (I have often 
seen them on my sprayed plants when they were less than one- 
fourth millimeter in diameter) , their color difference makes them 
conspicuous. Do not confuse with stigmonose. 




Fig. 217. — Distortion of bean leaves due to Bacterium phaseoli. From the 
same series of plants as Fig. 216, but the leaves were very ntnall tchen sprayed 
and most of the stomata were not open. Infection confined principally to the 
veins. 

In seasons of exceptional dewfall or rain the entire crop either 
of bush beans or of lima beans may be destroyed, especially in 
case of susceptible varieties. 

The geographical distribution of this disease is unknown. 
It occurs in many parts of the United States, probably in every 
state in the Union, but little is known concerning its occurrence 
in other parts of the world. Delacroix once reported it from 
France, but subsequently decided that his disease was different. 
I am inclined to think, since seeing his dried material and making 
sections of it (cultures failed), that his first conclusion is the cor- 
rect one, and that it does occur in France. I looked for it in 



THE BEAN BLIGHT: TYPE 



285 




vain, however, in the Paris markets in 1913. The same year 
at the International Exposition in Milan in the exhibit of the 
French Mycological Society I saw a yellow bacterial culture on 
slant agar, marked "La Graisse, " which was, perhaps, Bac- 
ierium phaseoli. It was made by Dr. T. A. Cordier of Rheims. 
The disease has been reported from South Russia by Spieshnev 
and from Japan by Arata Ideta who says : "This disease heavily 
damaged in Ishikari, Hokkaido, in 1903." Reinking has recently 
reported it from the Philippines 
{Phytopathology, vol. 9, 1919, p. 
131) where it is said to be "common 
and destructive." Miss Doidge 
uTites me (1919) that it is quite 
common in South Africa. 

Cause. — This disease is due to 
Bacterium phaseoli EFS. This is a 
yellow, non-viscid or slightly viscid, 
motile, polar flagellate (Fig. 227), 
non-capsulate, non-sporiferous. Gram 
negative, liquefying (both gelatin 
and L6 filer's blood serum), aerobic, 
non-gas-forming, non-nitrate-reduc- 
ing, starch-destroying, dry-air toler- 
ant, sunlight-sensitive (Fig. 228), 
frost-sensitive (in bouillon), single, 
clumping, catenulate, or filamentous 
schizomycete which grows on agar- 
poured plates in the form of small 
circular, or nearly circular, smooth, pale yellow, entire-margined 
colonies, becoming deeper yellow with age (denser and deeper 
yellow than those of Bacterium ynalvacearum) and then often 
pale ringed. Gelatin and Loffler's solidified blood serum are 
liquefied rather freely. Milk is curdled by means of a lab fer- 
ment, tyrosin being formed. The first evidence of curdling is the 
appearance of a shallow layer of clear whey above the slowly 
settling mobile curd. No acid is formed in milk. The organism 
resists drying. It grows feebly in Cohn's solution and in Uschin- 
sky's solution. The thermal death-point is approximately 50°C. 



Fig. 218.— Earliest stage of 
visible bean pod spot. Two in- 
fections each central through a 
single stoma. Plant sprayed 
Dec. 14 1914, and confined for 
19 hours in a roomy cage. 
Photographed Dec. 22. X 10. 
The organism used was a 72- 
hour agar-streak culture of the 
Idaho bean germ after reisola- 
tion from a plant successfully 
inoculated on Sept. 23. 



286 



BACTERIAL DISEASES OF PLANTS 




Fig. 219. — Bean blight due to Bacterium phaseoli: Pod sprayed December 14, 
1914, with a pure-culture suspension in water. Plants exposed 19 hours in the 
inoculation cage. This was one of the largest spots on a half-grown pod, most 
were decidedly smaller. Its regularity indicates a single central stomatal in- 
fection. There is a drop of yellowish bacterial ooze in the center of the spot. 
Variety. Extra Early Refugee. Planar enlargement by James F. Brewer. Photo- 
graphed December 26, 1914. X 6 circa. 



THE BEAN BLIGHT: CAUSE 



287 



(recent tests in +15 peptone beef bouillon). 
The Idaho organism was dead after 5 months 
on gelatin at 16° — 20°C. It is less sensitive 
to sunlight than Bacterium malvacearum. 

On a variety of culture media Bacterium 
phaseoli is closely like Bacterium campestre 
(See No. II) but the two organisms are not 
identical, as shown by the failure of repeated 
cross-inoculations (cabbage bacterium on 
beans and bean bacterium on cabbages), but 
our present means of separating the two 
forms culturally is insufficient. It is also cul- 
turally much like Bacterium citri, but with a 
virulent strain I failed to obtain any scabs on 
Citrus decumana (about 60 young seedlings). 
The student, therefore, who has opportunity 
might direct his attention to comparative 
studies of the yellow organisms of this group 
in the hope of finding additional cultural differ- 
ences by the use of new media. But in any 
event. Bacterium phaseoli belongs with Bac- 
terium campestre, Bacterium hyacinthi, Bacter- 
ium vascularum, Bacterium pruni, Bacterium 
malvacearum (No. X), Bacterium citri, and 
Bacterium translucens in a closely related 
kinship. 

Technic. — Isolations are easy, owing to the 
abundance and viabilit}^ of the bacteria. 
Only such ordinary precautions are necessary 
as have been described in detail for other dis- 
eases in this book, both earlier and later. 

Bacterium phaseoli is a good organism to 
work with because it does not lose virulence 
readily and plants for inoculation are quickly 
available at all times of the year. As ma- 
terial for experiment, both lima beans and bush 
beans ma}^ be used. Thej^ can be infected 
from germination almost to maturity, but the 








Fig. 220.— Bean 
pod sprayed with 
Bacterium phaseoli 
and kept for 26 
hours in an inocu- 
lation cage. The 
organism used was 
cultivated from the 
pod shown in Fig. 
22 1. Time, 14 
davs. 1914. 



288 



15ACTERIAL DISEASES OF PLANTS 



various organs are most susceptible during early stages of 
growth. The beans should be planted 6 or 8 weeks before they 
are needed and must be shifted occasionally if the pots are 
small, so that they will continue to develop rapidly. It is best 
to plant them in 6-inch pots, shifting to 8-inch or 10-inch pots 





Fig. 221. Fig. 222. 

Fig. 221. — Enlarged base of a bean pod showing translucent exudate, due 
to Bacterium phaseoli. Pod received from Idaho in 1914. The organism isolated 
from it served for a long series of infections. It is still infectious (1920). 

Fig. 222. — Bacterium phaseoli on bean. Same series of stomatal infections 
as Fig. 220, but later. Spots now large, somewhat depressed and exuding bacteria 
freely from their center. Time, 19 days. 



as they require it. They are very easy to grow and need only 
ordinary care. There may be 2 to 4 plants in a pot, if plenty of 
good soil is used. 

The inoculations should be both by needle puncture and by 
spraying since the disease is readily transmitted through the 



THE BEAN BLIGHT: TECHNIC 



289 



stomata. Indeed, it is one of the most convenient diseases for 
studying stomatal infections. 

The needle-pricks should be made on young pods, soft stems, 
and various parts of the immature leaf (petiole, petiolule, veins 
and parenchyma). For comparison try also needle inoculations 
in full-grown leaves, stems and fruits. 

For the spraying experiments, plants a foot high, bearing 
mature, immature and undeveloped leaves, should be selected, 




Fig. 223. — Bacterial cavity in a bean leaf 5 days after spraying on a pure 
culture of Bacterium phaseoli. Tissue not yet collapsed. Stomatal infection 
through S, S. 

SO as to observe the modifying influence on the disease of age of 
tissues. The leaves should be free from insects, fungi, and con- 
fusing spots of any sort. The plants should be atomized with 
water holding in suspension bacteria from young (3-day) agar- 
streak cultures until the surface is covered with small drops of 
the cloudy fluid. Then the cages should be closed tightly and 
protected from the light. They should be examined every few 
hours throughout the daytime to see that the drops have not 
evaporated. To insure this favorable persistence of moisture 

19 



290 



BACTERIAL DISEASES OF PLANTS 



on the plants it is necessary usually to wet down thoroughly the 
interior of the cage and the earth under and around it in advance 
of the inoculations. If moisture does not hold on the leaves they 
must be sprayed again, and the earth wet down as before. 
Watch carefully, for if the plants dry off speedily and remain 
dry your experiment may not succeed. The plants should be 
left in the cages only as long as necessary to secure numerous 
infections. The writer does not know the minimum time. 




Fig. 224. — Bacterium phaseoli: A detail from Fig. 223, showing the bacteria 

more distinctly. 



Perhaps you can determine it. He has had striking results from 
26- and 30-hour exposures, equivalent to a dewy night followed 
by a misty day. Probably a very considerably shorter period 
of exposure would suffice. In default of cages, clean barrels 
or boxes may be turned over the plants, or they may be cov- 
ered with a tent-cloth or an oilcloth stretched over a frame. 
Tent-cloths require very frequent wetting. 

King of the Garden (lima), Mexican tree bean, Green Flageo- 



THE BEAN BLIGHT: TECHNIC 



291 



let, and Red Valentine are susceptible varieties, also many others 
including Early Refugee, reported as resistant. 

Infections appear sooner and the disease progresses faster 
in warm weather than in cool weather. If there are facilities, 
two sets of spray inoculations may be identical in all respects 
except as to temperature, i.e., one in a greenhouse at 30° to 
35°C., the other in a house at 18° to 20°C. 

Determine 

For the organism. Morphology. — Size in microns. Con- 
ditions under which chains and filaments occur. Absence of 







Fig. 225. — Bacterial invasion in outer tissues of a bean pod. Epidermis lifted 
by bacterial pressure in the stomatal region. Intercellular spaces occupied. A 
stomatal infection obtained by spraying. Time, 12 daj's. 1914. 

endospores. Number and attachment of flagella. Conditions 
leading to the formation of clumped masses (pseudozoogloeae). 
Do involution forms occur? Are the rods ever curved or larger 
at one end than at the other (see No. II)? Do capsules occur? 
Cultural Characters. — Determine appearance of colonies on 
thin-sown agar plates (figs. 229, 230) ; behavior in agar stabs and 



292 BACTERIAL DISEASES OF PLANTS 

Streaks; growth on gelatin; in L6 filer's solidified blood serum, 
in Dunham's solution (see fig. 92.4). Does it cloud Dunham's 
solution? Study behavior on potato. Hold the potatoes for 
6 weeks and then test the substratum for destruction of starch, 
mashing the cylinders in an abundance of distilled water (50 
cc.) to which should then be added 2 cc. of alcohol iodine. Com- 
pare with a mashed check cylinder of the steamed potato and 




'4* 




1 




Fig. 226. — Cavities in a bean pod (outer part) due to introducing Bacterium 
phaseoli by needle-pricks. Inoculated by the writer in 1897. 

with cultures of Nos. II and III. Has the potato lost its firm- 
ness? Why? The progressive enzymic destruction of potato 
starch (change from translucent, lustrous, bluish white to a dead 
opaque, white) may be watched from day to day if streaks are 
made in test tubes on slant starch jelly. (For its preparation see 
"Bacteria in Relation to Plant Diseases," Vol. I, pp. 50, 196.) 
Determine action on milk and litmus milk. After some weeks' 



THE BEAN BLIGHT: CULTURAL CHARACTERS 293 

growth in milk, filter the translucent fluid through coarse ster- 
ile paper or sterile cheesecloth and divide it into two equal 
portions a and 6: heat a for 20 minutes in the water-bath at 80°C. 
and pour it into a test tube of sterile milk; heat b for 20 minutes 
at 50°C. (to kill the bacteria), streak on potato copiously to de- 
termine that they have been killed, and pour into another test 
tube of sterile milk. Add a small crystal of thymol to each 
tube by means of sterile forceps. Watch the two closely for 
the next 48 hours. The milk which received b should curdle in 
the absence of the living bacteria, that which received a should 
not curdle. How do you account for the difference? Observe 
the tubes of inoculated litmus milk carefully from time to 
time. Is there ever anj^ acid reaction? Do not be deceived by 
appearance of tubes when held up to the light; all litmus solu- 
tions are red by transmitted light. Examine and decide only 
by reflected light. 

Study growth in peptone bouillon; ditto in nitrate bouillon. 
Are nitrates reduced? 

Behavior in Cohn's solution — in Uschinsky's solution. 

Growth in peptone water in fermentation tubes with various 
sugars and alcohols. Compare with Bacterium campestre 
and with Bacterium malvacearum, testing as many carbon com- 
pounds as possible. 

Stud}"- effect of freezing (4-hour tube cultures in + 15 peptone 
beef bouillon in salt and pounded ice for one hour). In 1919 we 
tried five isolations with the following per cents of killing: 
Idaho, original stock, 87; Idaho, G. H., 98; Michigan, 99+ ; 
New York, 89; Washington, D. C., 99+ ; Maryland, 99 + . Read 
Science N. S., vol. XXI, No. 535, March 31, 1905, pp. 481-483. 

Is litmus reduced? Is indol formed? Ammonia? Carbon 
disulphide? Invertase? Catalase? Pour some fresh hydrogen 
peroxide into an old potato culture and observe the result. 
What do you conclude as to the nature of the reaction? What 
other ferments are formed? Nature and solvents of the yellow 
pigment? Is it a lipochrome? 

N on- nutritional Environ7nent. — Reaction to heat, frost, sun- 
Ught, dry air, germicides. Behavior on media (use a variety) 
in non-respirable gases — carbon dioxide, hydrogen, nitrogen. 



294 BACTERIAL DISEASES OF PLANTS 

For the disease. Signs. — After needle-prick inoculations 
on young leaves, stems and pods, how long to first appearance 
of the disease? Rate of progress of the infection. 

On sprayed plants how long before faint pale green dots can 
be detected on the leaves (use hand lens and transmitted light, 
examining every day)? How many additional days are re- 
quired for these stomatal infections to become visible to the 
naked eye by reflected light? How long before they become 
conspicuous, forming definite, coalescing sunken spots? Have 
you observed retention of the leaf-green around the bacterial 

spots and discharge of it in other 
• parts of the leaf? How do you 

account for this? 
*^ * Make similar observations on 

sprayed pods as to time of first 
it appearance of spots, rate of prog- 

ress, etc. Temperature has some- 
, ^ thing to do with this, therefore 

f . '. \ "■ ' keep thermometric records. 

' ^- " " ^ Describe the disease minutely, 

A ,4^ including its effect upon the pods 

* ,^r^ and its general effect upon the 

./ plant. Are the roots ever dis- 

Ci- eased? Are the pods dwarfed? 

Fig. 227.— Flagellate rods of Histology .Study the manner 

Bacterium phaseoli. Idaho isola- of entrance of the bacteria into 
tion. Stained by van Ermengem's ^j^e bean-pod, employing Very 

silver nitrate method. Photoniic- ^ t •, i 

rograph by the writer. X 1000. ^O^ing spotS. Is it always Stom- 

atal? Is it generally so? In well- 
developed spots on the pods, determine the manner of extrusion 
of the bacteria. Is it always stomatal or may it be through rifts 
in the tissues? (Sometimes the damp chamber may be of assis- 
tance in determining this.) Still using the pods, make cross- 
sections (free-hand and on the microtome) of spots in various 
stages of development to determine what tissues are invaded by 
the bacteria and how this invasion takes place. Make perma- 
nent preparations. Young pods will cut much better than old 
ones, but tissues of all parts, and of all plants, for that matter, will 



THE BEAN BLIGHT: HISTOLOGY 



295 



cut better if the air is pumped out of them when they are first 
put into the acid-alcohol or other fixative. 

Do the same things with the leaf spots. Is there any 
increase in the number of chloroplasts in cells surrounding the 
leaf spots? To what is the russet or rusty-red phenomenon due? 
What becomes of the cell-walls? How is the middle lamella dis- 
posed of? Ziehl's carbol fuchsin is a good stain. 

Is the tissue killed in advance of bacterial occupation? 
What are your reasons for thinking so? 




Fig. 228. — Agar-poured plates of Bacterium phaseoli (Idaho isolation). Inner 
one-half of each exposed to bright sunlight on ice: a, 2 minutes; b, 5 minutes, and 
then incubated for 6 days. Much less sensitive than Bacterium malvacearum. 
Compare with Fig. 249. Photographed June 16, 1915. >2 nat. size. 



Does the organism frequently penetrate the seed coats? 

On stem-inoculated plants how far can you trace the bacteria 
in the vascular bundles? Is the phloem attacked? 

Variability. — Under field conditions different varieties of 
beans show marked differences in susceptibility. If the student 
has opportunity he should study variability in the field and 
garden, being always on the lookout for resistant varieties 
and hardy individuals in susceptible varieties. Make careful 
(and legible) pen notes of what you have seen. 

Transmission. — We know nothing concerning living carriers 
of the organism but, owing to the free oozing of the bacteria 
to the surface, any bird or insect might act as a carrier from 
diseased to healthy surfaces, as in case of fire-blight of apple 



296 BACTERIAL DISEASES OF PLANTS 

and pear (No. XII), the only subsequent agent necessary 
being rain or dew. That a considerably greater number of 
hours of continuous moisture (than under experimental, fresh- 
culture, hothouse conditions) is necessary to insure infection 
under natural field conditions, is shown, I think, by Halsted's 
observation that in a New Jersey bean field nine-tenths of the 
infections were on the west side of the pods, i.e., on that side 




Fig. 22\).—Bticteriiun phaseoli: Buried and surface colonies on an agar poured 
plate. Idaho isolation. Plate poured August 24, 1914. Photographed August 
28. X 10. Oblique lighting. 

likely to remain damp longest because shaded from the morning 
sun. (In this connection read comment on black spot of the 
plum in "Bacteria in Relation to Plant Diseases," Vol. II, p. 62). 
Can you verify Dr. Halsted's observations? Do you think 
that what he saw could have been due to driving rains from the 
west? In that case the infection should sometimes occur most 
abundantly on the east side of the pods. Watch for this. 

Barlow proved by transfers to culture media that Bacterium 



THE BEAN BLIGHT: TRANSMISSION ^ 297 

phaseoli can live over winter on naturally infected seeds (kept 
both in the pods and in sterile test tubes) and also determined 
that such seeds were not injured beyond the power to germinate 
and grow, but does not state that he traced the disease on into 
the seedlings derived from such seeds. 

It has not yet been proved experimentally that this disease 
is co7nmonly carried on the seed, unless we may assume that 
Edgerton has done so, but such I believe to be the case (read 
what is said under Nos. II and III, and respecting Rathay's 
Disease of Orchard Grass in "Bacteria in Relation to Plant 
Diseases," Vol. Ill, p. 160, and make all the observations and 
experiments you can). My reasons for this belief in addition to 
Barlow's statement, and Edgerton's, are the facts drawn from 
my own observations that the bean bacterium is not very 
sensitive to dry air, and that it may pass entirely through the 
walls of the pericarp and infect the seeds without destroying 
them, i.e., as they are approaching maturity. Such seeds, 
which are usually more or less distorted, should be saved in 
large numbers in sterile tubes and tested from time to time 
through the autumn, winter, and spring, to determine: (1) 
whether Bacterium phaseoli can be cultivated from many of 
them; and (2) especially whether seedlings grown from such 
seeds (in autoclaved soil and watered with boiled water) do 
commonly become infected. 

Here is a definite, interesting, practical problem, easy of 
solution — given a bean field containing an abundance of the 
disease and a student with some aptitude for research. Five 
hundred or a thousand diseased bean pods would not be too 
many to save for such an experiment. A little preliminary 
observation will determine what pods should be selected, i.e., 
how badly diseased they must be externally to warrant belief 
that the pericarp has been perforated. Bean cotyledons are 
often distorted and bear rusty spots when they emerge from the 
soil. Is any part of this phenomenon the disease in question, 
or is it all due to fungous infection? 

If Barlow's conclusions can be generalized, as seems prob- 
able, we shall have a very simple and practical way of dealing 
with this disease. 



298 



BACTERIAL DISEASES OF PLANTS 






Fig. 230. — Buried and surface colonies of Bacterium phaseoli (Idaho isolation; 
in a thin-sown +15 peptone-beef-agar poured plate. The smooth, wet-shining, 
yellow, surface colonies show internal markings by direct transmitted light. Plate 
poured April 11, 1919. Photographed April 18. X 10. Temperature 25°C. 



THE BEAN BLIGHT: TRANSMISSION 299 

If the organism is generally carried on the seed, then seed 
treatments are in order, and these should be made anyway, 
until it is known that they are of no avail. In this connection 
see Part I, page 69. 

Another important subject for field study is the discovery or 
production of good resistant varieties. The subject is very 
hopeful if the right persons take hold of it. 




Fig. 230*. — Agar plate of Bad. phaseoli showing a common form of colony 
referred to in the text. Organism isolated by Florence Hedges in 1917 from a 
Maryland bean pod and used many times for successful inoculations. Plate 
poured May 22, 1920, and photographed June 4. X 5. 

LITERATURE 

Beach: Bull. 48, X. 8., X. Y. Agr. Exp. Sta. (Geneva) 1892. 

Smith: Proc. Am. Assoc. Adv. Sci., Vol. XL VI, 1897, pp. 288- 
290. Species here named Bacillus phaseoli. 

Smith: Bull. 28, Div. Veg. Phys. and Path. U. S. Dept. of 
Agric, 1901. 

Halsted: Bull. 151, X. J. Agr. Exp. Sta., 1901. 

Barlow: Bull. 136, Ontario Agr. Exp. Sta., 1904. 

Sackett: Bull. 138, Colorado Agr. Exp. Sta., 1909. 

Edgerton and Morelaxd : The bean blight and preservation 
and treatment of bean seed. La. Agr. Expt. Sta. Bull. 139, 
Baton Rouge, La. 1913. 

Rapp : Aged bean seed, a control for bacterial blight of beans. 
Science, n. s., Vol. L, Dec. 19, 1919, p. 568. 

See also ''Bacteria in Relation to Plant Diseases," Vol. I, 
Fig. 62; Vol. II, Fig. 15 and pi. 17 (Fig. 3). 



IX. MCCULLOCH'S CAULIFLOWER SPOT 

Type. — This is a disease of cauliflower and cabbage, char- 
acterized by a copious stomatal blotching, spotting or specking 
of the green leaves, both on the veins and in the parenchyma. 
When the midrib and the veins are attacked early and seriously, 
the leaves become variously puckered and distorted (see also 
Nos. VIII and XI). Seen by reflected light, the spots are at 
first water-soaked, then brownish to purplish gray, but by trans- 
mitted light they appear thin and almost colorless in the center, 
with a dark border. The spots on cabbage are darker than those 
on the cauliflower. The individual spots, which occur in great 
numbers, are usually small (mere points to areas 1 to 3 milli- 
meters in diameter, seldom larger), and are circular when quite 
small but soon become more or less irregularly angled, owing 
to limiting veins. By coalescence, elongated, irregular, and 
much larger spots occur, making the leaves look ragged. The 
badly spotted leaves also turn yellow and fall off (3 to 5 weeks 
after infection). 

The disease was not observed naturally infecting the bleached 
flowering parts of the cauliflower in the Virginia material, nor 
was it obtained by spraying such parts, except sparingly on 
some of the larger flowerstalks, but later was obtained on cauli- 
flower heads from Florida and produced on the cauliflower heads 
in one of our hothouses by pure culture inoculations. Probably 
the stomata do not function as readily on the bleached parts as 
on the green parts and consequently are less likely to be entered 
by the bacteria. 

All of the infections, so far as observed, are stomatal, each 
small round spot having a single stoma in its center, below 
which is a bacterial pocket. By spraying water-suspensions of 
young agar-streak cultures upon cauliflower plants and cabbage 
plants, numerous infections were obtained (Fig. 231) but mostly 
on the under surface of the leaves. When the infectious spray 
was confined to the upper surface of the leaves very few spots 

300 



MCCULLOCH S CAULIFLOWER SPOT: TYPE 



301 



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Fig. 23L — Cauliflower leaves attacked and spotted by, Baciermni macuU- 
colum, McCulloch's cauliflower parasite. A pure-culture inoculation. Time, 
13 days (May 19 to June 2, 1909). The infections are stomatal. 



302 



BACTERIAL DISEASES OF PLANTS 



were obtained. Time of day might make a difference. The old 
and the very young leaves appear to be partially or wholly 
immune (see observations under Nos. VIII and X). 




Fig. 232. — Spotted cauliflower head from Sanford, Florida, April 3, 1916. Gray 
to black discoloration of the epidermis and deeper tissues of the inflorescence. 
Plates were poured by Miss McCuUoch on April 4 and Bacterium maculicolum 
was obtained and used for successful inoculations. 

The period of incubation is short, numerous spots being 
visible within 3 to 6 days from the time of spraying, and always 
beginning, as in the spots on the naturally infected field cauli- 
flowers, in the substomatic chamber. 



MCCULLOCH S CAULIFLOWER SPOT: TYPE 



303 




Fig. 233. — Peptone-beef-agar poured plate ( + 15) of Bacterium maculicolum 
plated April 28, 1916, from spot on a cauliflower leaf which was inoculated April 24 
with the Sanford, Florida, organism. Photographed May 2, with oblique trans- 
mitted light for internal colony markings, the surface being smooth. X 10. 



304 BACTERIAL DISEASES OF PLANTS 

Little is known regarding the geographical distribution of 
this disease. It was first received at the Laboratory of Plant 
Pathology in Washington on cauliflower leaves from Southeast- 
ern Virginia in April and May, 1909. Subsequently (1912) 
something on cauliflower resembling it was received from Pal- 
metto, Florida, and Miss McCulloch succeeded in isolating an 
organism that culturally seemed right but no inoculations were 
made. Later (1916) from cauliflower heads grown at Sanford, 
Florida (Fig. 232), the organism was plated out (Fig. 233) and with 
it pure culture inoculations were obtained (Fig. 234). In 1918 it 
was received from Prof. H. H. Whetzel on cauliflower leaves 
collected in Ithaca, New York (Fig. 235). At first Prof. Whetzel 
thought that the disease was only a peculiar form of the black 
rot (see No. II) but since the leaves were without marginal 
infection and the petioles were not diseased, and the spots did not 
yield any yellow organism, he sent the leaves on to me to know 
what it was. Miss McCulloch obtained from it her Bacterium 
maculicolum, with pure cultures of w^hich she secured typical 
infections on cauliflower in one of our houses, and from such 
infections again obtained in pure culture typical colonies of the 
organism (Fig. 236). In April, 1919 we received it from the New 
Orleans market (courtesy of Lex R. Hessler) on cauliflowers said 
to have been grown in California. 

This disease probably occurs also in Australia, that is, in 
1900 I received specimens of cauliflower leaves and cabbage 
leaves from Prof. D. Mc Alpine in Melbourne, bacterially spotted 
with what I now believe to be this disease. His letter states 
that it is the cause of serious damage. 

The conclusion respecting occurrence of the disease in cab- 
bages depends upon artificial inoculations made in Washington 
in 1909 and 1910. 

We owe our knowledge of this disease to the researches of 
Lucia McCulloch, carried on in my laboratory during the years 
1909, 1910, 1911 and later. No one else appears to have written 
upon it. 

Cause. — The cauliflower leaf-spot is due to Bacterium maculi- 
colum McCulloch. This is a moderately growing, white, motile 
(even after 4 months in beef bouillon at 0.5°C. to 1.5°C.), 1-5 



MCCULLOCH S CAULIFLOWER SPOT: CAUSE 



305 




Fig. 234. — Bacteriimi maculicolum: Cross-section of cauliflower inflorescence 
(buds) showing depth of black spotting. From a pure-culture inoculation made 
November 20, 1916, using the Florida organism. Collected and fixed December 7. 
Slide 131055, second section, upper row. Carbol fuchsin stain. 16 mm., 4 oc. 
50 bellows. 

20 



306 BACTERIAL DISEASES OF PLANTS 

polar flagellate (Fig. 237), non-sporiferoiis, Gram negative, non- 
acid-fast, liquefying (gelatin, not Loffler's blood serum), milk- 
clearing (by a lab ferment with formation of tyrosin), non-gas- 
forming and aerobic (in fermentation tubes in peptone water 
with dextrose, saccharose, lactose, maltose, glycerin ormannit), 
non-nitrate-reducing, green fluorescent (a pale yellowish-green 
stain in milk, peptone beef bouillon, peptone beef agar, peptone 
beef gelatin, Fermi's solution and Uschinsky's solution), alkali- 
tolerant (NaOH down to -25 in beef bouillon), acid tolerant (in 
bouillon up to +34 for oxalic acid, and +36 for malic acid and 
citric acid), sodium chlorid-sensitive (beyond 2 per cent — at 
4 per cent in bouillon motility ceases, and at 5 per cent there 
is scarcely any growth); chloroform-tolerant; grows below 0°C. 
but is injured by freezing in bouillon (70 to 90 per cent killed) ^, 
dry-air sensitive (very), sunlight-sensitive (very), heat-sensitive 
(very), short rod-shaped, catenulate (on agar and in 4 per cent. 
NaCl bouillon) or filamentous schizomycete (0.8 to 0.9^ in 
diameter), growing on +15 peptone beef agar plates in form of 
round, smooth, flat, shining, translucent, opalescent, white, 
entire-margined colonies, becoming 1 to 3 mm. in diameter in 
3 or 4 days at 23°C., at which time or earlier the inner structure 
is coarsely granular and reticulate under the hand-lens. Later 
the inner structure becomes finely granular and the reticulations 
disappear. . In thin-sown plates at the end of 7 days the surface 
colonies are 6 to 8 mm. in diameter, and after 15 days 12 to 
15 mm. in diameter. With age the colonies become dull or dirty 
white, and slightly irregular in shape, with undulate or faintly 
crinkled margins into which run indistinct radiating lines. The 
buried colonies are small and lens-shaped. 

On +10 nutrient beef-peptone gelatin plates, after 3 days 
at 17° to 18°C., the well-isolated colonies vary from mere points 
to round growths 2 mm. in diameter. The gelatin is liquefied 

^ The statement on p. 13 of Bull. No. 225, Bureau of Plant Industry, is erroneous. 

Fig. 235. — One of a number of spotted and blotched New York cauliflower 
leaves received from Prof. H. H. Whetzel in the fall of 1918. The leaves were 
yellowish and the spots pale green to black. These gave an organism culturally 
the same as Bacterium macuHcolurn McC, and this produced the characteristic 
lesions when sprayed upon cauliflowers in one of our houses, and from one of these 
Fig. 236 was obtained. Good infections were also obtained with it in Feb., 1920. 



Mcculloch's cauliflower spot: cause 



307 




Fig. 235. 



308 BACTERIAL DISEASES OF PLANTS 

in cup-like hollows. The margin of the smaller colonies is 
entire, that of the larger ones is fimbriate. Thickly sown plates 
are entirely liquefied in 2 days at 15° to 16°C. 

Peptonized +15 beef bouillon, if inoculated from young 
vigorous bouillon cultures, clouds thinly in 6 hours and moder- 
ately to heavily in 24 hours at 24° to 25°C. The growth is best 
at the surface, where a fragile white pellicle forms. There is 
no rim, and no pseudozoogloeae are formed. In two days there 
are heavy clouds and a flocculent white precipitate which is 
slimy and finally viscid. The medium becomes slightly greenish 
and small crystals appear in the sediment. 

In acid bouillons, pseudozoogloeae may occur and the rods 
become very short (spheroidal). 

In —17 beef bouillon both filaments and involution forms 
were seen at the end of 2 weeks. 

A fragile white pellicle forms also on Fermi's solution and 
Uschinsky's solution, and pseudozoogloeae occur in the latter. 

In Cohn's solution it grows without green fluorescence, rim, 
or pellicle, but with the formation of large feathery crystals. 

The organism blues litmus milk in well-defined strata 
from the top downward; no acid is formed, the cultures being 
dark blue by reflected light even after 6 months. 

The color-changes in milk likewise proceed from the top 
downward in definite layers. In 15 to 20 days the whole tube is 
yellow with a greenish tinge; it is translucent but without de- 
struction of the fat or separation of whey and curd. In 4 months 
when evaporated from 10 cc. to 5 cc. the milk is reddish brown 
and rather thick. 

The organism is a cool-weather parasite. Its optimum 
temperature is 24° to 25°C., its maximum (in bouillon and on 
agar) is 29°C., and its minimum below 0°C. Of 20 bouillon 
cultures exposed for 10 minutes at 47°C. none grew; of 12 bouil- 
lon cultures exposed for 10 minutes at 46°C., one clouded after 
11 days; exposure of bouillon for 10 minutes at 45°C. killed the 
bacteria in some of the tubes (less than one-half) and retarded 
development in the others 3 to 5 days, i.e., many were killed; 
finally, exposure in beef bouillon for 3^2 days at 33° to 36°C. 
destroyed the organism, i.e., prevented subsequent clouding at 



Mcculloch's cauliflower spot: cause 



309 




Fig. 236. — Surface and buried colonies of Bacterium maculicolum plated 
from the New York cauliflower Three buried colonies coming to the surface. 
Photographed by oblique transmitted light to show internal colony markings. 
Plate made from a spot on an inoculated plant. X 10. 



310 BACTERIAL DISEASES OF PLANTS 

optimum temperatures. No growth could be obtained in the 
plant or on agar or in bouillon at or above 29°C. (84°F.). 

The organism is rather long-Uved on media but loses viru- 
lence readily. It stains deeply with carbol fuchsin and by 
amyl Gram. There is a feeble production of ammonia, indol and 
hydrogen sulphide. A few cultures in litmus milk showed a 
slight reduction of the litmus at the bottom. 

It is extremely sensitive to dry air and to sunlight. Four 
minutes exposure to direct sunshine killed all organisms in the 
insolated half of the thin-sown agar plates, exposed bottom 
up on ice, although the colonies developed as usual on the covered 
half of each plate. Taken from young, well-clouded bouillon 
cultures, and dried in the dark at 22° to 25°C. on cover glasses 
which were then dropped into suitable bouillon, the bacteria were 
dead on 75 per cent of the covers in 24 hours, on 90 per cent in 
48 hours, and on all at the end of 5 days. 

Technic. — The organism causing this disease grows readily 
in +15 agar-poured plates when the temperature is under 29°C. 
(not at all at or above this temperature)^ and there are no 
difficulties in the way of isolation, other than surface con- 
taminations, which are held in check, more or less, by short ex- 
posures (20 to 30 seconds) in 1 : 1000 mercuric chlorid water, 
after which the spots are crushed in bouillon for the poured 
plates. 

^ I felt so sceptical about 29°C. as the maximum temperature that in January, 
1919, I asked Miss McCulloch to do over for me this part of her work, which she 
did with the following results: 

Tests of Bacterium maculicolum for maximum temperature. Isolations used: 
f Sanford, Florida, Col. 2 of April, 1916. 
' Floral Stalk, Col. 1, December, 1916. 
Leaf, Col. 2, December, 1916. 
Mid-rib, Col. 6, March, 1917. 
I Ithaca, New York, Col. 3, November, 1918. 
I Greenhouse, Cols. 10, 11, 13 of January, 1919. 

(1) 18-25°C. 
All of the above isolations clouded +15 peptone beef bouillon moder- 
ately in 16-18 hours. 

(2) 28.5-30°C. (Incubator heated by electricity and as workmen were chang- 
ing wires the current was sometimes off.) 

"A" strains, which have been in artificial media from two to three 
years, clouded slightly in 18 hours. 



Mcculloch's cauliflower spot: technic 311 

For the inoculations, growing cabbage and cauliflower 
plants 6 to 12 inches high may be used. These, preferably, 
should be made in infection cages, by spraying the under surface 
of the leaves with water containing suspensions of young agar- 
streak cultures. If proper conditions are obtained, good in- 
fections in large numbers should be available for study by the 
end of the first week. By "proper conditions" are meant: 
(1) moist conditions for 48 hours; (2) growing plants; (3) 
temperatures under 29°C. (84°F.); and (4) use of bacterial 
cultures which have not lost their virulence or become attenu- 
ated by harmful laboratory conditions. Miss McCulloch 
several times obtained numerous infections on both cauliflower 
and cabbage plants by spraying, but none in hot weather and 
none or few with the descendants of old cultures. It is possible, 
however, that some of her isolations may represent feeble strains 
of this parasite. 

Under our rather dry hothouse conditions secondary in- 
fections were not observed. That is, the infections were 
limited to the leaves actually sprayed. 

Determine 

For the organism. Morphology. — Size in microns. Ab- 
sence of endospores (try heat and spore stains). Motility 
on the margin of a hanging drop. Persistence of motility 
in old cultures. Number and distribution of flagella (use 
van Ermengem's or Hugh Wilhams's method). Conditions 



"B" strains were not clouded in 18 hours. 
After three days there was faint clouding in "B"' strains. 
In six days the growth in "B" strains is less than in room temperature 
cultures in 18 hours. 

(3) 29.5-30.5°C. 

"A" strains were slightly clouded in 24 hours. 

"B" strains not clouded. 

"B" strains not clouded in four days. 

(4) 30-31°C. 

"A" strains clouded faintly in 24 hours. 
"B" strains not clouded in four days. 



312 BACTERIAL DISEASES OF PLANTS 

under which chains and filaments are formed. Invohition 
forms. Under what conditions do they occur? 

Cultural Characters. — Behavior on thin-sown agar-poured 
plates. Ditto in agar-streak cultures and stab cultures. Char- 
acter of growths on thin-sown gelatin-poured plates. Ditto 
in stab cultures and streak cultures on gelatin. Growth in 
streaks on Loffler's solidified blood serum. Behavior on potato. 
Growth in milk and litmus milk (examine every day). Growth 
in synthetic media — Cohn's solution, Fermi's solution, Uschin- 
sky's solution, etc. Action in peptone water on various sugars 
and alcohols in fermentation tubes. Is any gas produced? 
Is there ever any clouding of the closed end? Is any acid 
formed in the open end? Test in appropriate media for pro- 
duction of indol, ammonia, hydrogen sulphid, amylase, lab 
ferment, proteolytic ferment. 

Non-nutritional Environment. — Thermal death-point (begin 
with 45°C.). Maximum temperature for growth (try first in 
-|-15 peptone beef bouillon at 30°C.). Optimum temperature 
for growth (use measured loops into bouillon and examine every 
three hours). Lowest temperature at which growth occurs 
(try first at 1°C. and continue experiment for six weeks). 
Effect of dry air (using young bouillon cultures first). Effect 
of insolation (expose on ice to a bright sun for 5 minutes and 
hold half of each plate covered as a check). Effect of freezing 
(salt and pounded ice). Behavior in salted bouillons (try 
5 per cent first) . Behavior in bouillon over chloroform. Tolera- 
tion of sodium hydroxide in bouillon (begin with — 22) . Tolera- 
tion of organic acids in bouillon — tartaric, citric, malic, etc. 
(begin with +30). Action of fungicides. 

For the disease. Signs. — Describe early, middle and 
late stages of the disease and determine how long it takes to 
produce these conditions on sprayed plants at given tempera- 
tures. Make inoculation experiments at the same time in two 
houses (or places) having temperatures 10 degrees apart: 
one 20° to 25°C., the other at 30° to 35°C. Are Miss Mc- 
Culloch's statements as to impossibility of obtaining growth 
or infections at or above 29°C. entirely correct? In that case, 



I 



Mcculloch's cauliflower spot: signs 313 

where does the organism summer over? Can you obtain the 
disease on the bleached flowering parts? 

Histology. — Fix in Carnoy's solution pieces of cauliflower 
leaves and heads showing small spots, carry them through the 
paraffin-infiltration process, section on the microtome, stain and 
study. What are your conclusions regarding manner of infec- 



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Fig. 237. — Flagella of Bacterium macidicolum. Stained from a 2- day agar streak 
by van Ermengem's method. X 1000. 

tion and process of tissue destruction? Is the disease ever 
vascular? Is cellulose destroyed? 

Variability. — Nothing is known. 

Transmission. — Nothing is known 

LITERATURE 

Read McCulloch: ''A Spot Disease of Cauliflower." Bul- 
letin 225, Bureau of Plant Industry. Washington, D. C, 
Government Printing Office, 1911. The organism is described 
and named in this paper. 



X. THE ANGULAR LEAF-SPOT OF COTTON 

Type. — This disease is a common leaf-spot, twig-blight and 
boll-rot of cotton, comparable with the bean blight due to 
Bacterium phaseoli (No. VIII), the walnut blight due to Bac- 
terium juglandis, and in some ways also with the mulberry blight 
due to Bacterium mori (No. XI). 

Following Atkinson, the leaf-disease is commonly known 
as the angular leaf-spot. The stem-blight is known as "black- 
arm" or " gummosis," and the capsule spot as "boll-rot." These, 
however, are only different manifestations of one disease. 




%,*. 



Fig. 238. — Cotton leaves from Monetta, South Carolina, showing Atkinson's 
angular leaf spot, due to Bacterium malvacearum. Photographed in 1903. }4 
nat. size. 

The spots on the leaves were first described in 1891-92 by 
Geo. F. Atkinson who examined them under the microscope and 
detected bacteria in them. He isolated a micro-organism and 
made inoculations, but these were unsuccessful. 

Stedman ascribed the boll-rot to his green-fluorescent 
Bacillus gossypina, but on insufficient evidence. 

The writer was the first to reproduce the disease on leaves 
and bolls of healthy cotton plants (1900-1905) with the true 
parasite isolated from capsule-spots and leaf-spots. He was 

314 



THE ANGULAR LEAF-SPOT OF COTTON: TYPE 



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BACTERIAL DISEASES OF PLANTS 



also the one who proved the stem-blight known as black-arm 
and the angular leaf-spot to be due to the same organism (Wash- 
ington hothouse inoculations of 1905). The inclusion of black- 
arm in Mr. Orton's account of the bacterial cotton-blight 
("Sea Island Cotton: Its Culture, Improvement and Diseases," 
U. S. Dept. Agric, Farmers' Bulletin 302, 1907, pp. 41-42) 
was due to these experiments, some of which he had seen. 

The first indications of the disease are minute water-soaked 
spots. On the half-grown leaves these spots are chiefly between 
the veins and more or less limited by the latter, the result as 




Fig. 240. — Cotton leaves inoculated in spring of 1915, using a pure culture 
plated from the lower leaf of Fig. 239. Time, 32 days, cool house. Stomatal 
infection; veins also invaded. 



they enlarge being square or variously angled small spots (Fig. 
238) which are translucent at first, then brown. These spots 
by their number and their one-sided enlargement and coa- 
lescence may seriously injure the attacked leaves, greatly reduc- 
ing the amount of assimilating surface and causing them to 
become yellow and to fall early. The bracts and the veins or ribs 
of the leaf are also subject (Fig. 239). Contrary to Atkinson's 
supposition, infection often takes place very early, i.e., when the 



THE ANGULAR LEAF-SPOT OF COTTON: TYPE 317 

leaves are quite small, and then the disease is generally confined 
to the larger veins or ribs of the leaf (Figs. 240 and 241). 

The infections are largely stomatal and are easily induced 
by spraying on water suspensions of pure cultures (Fig. 242). 
Two to three weeks or more, depending on the temperature, are 
required for stomatal leaf infections to become plainly visible. 
Their interior is then as shown in Fig. 243, i.e., the tissues are 
disintegrating and full of bacteria. The shortest period I have 
observed for both the vein disease and the parenchyma spot is 
12 days. This was in very hot weather in July, 1915, using as 
source of infection potato sub-cultures from a "windowed" 
colony of the Arizona organism, cultivated from the leaf shown 
in Fig. 244. 

On the green bolls which are also attacked (Fig. 245), the 
water-soaked areas enlarge slowly or rapidly according to 
the age of the bolls and the weather conditions. If the bolls 
are small when attacked they drop off, and if large they become 
first green-spotted, then brown or black-spotted and sunken in 
the attacked parts, and the lint either fails to develop under 
the spots or becomes wet, brown-stained and rotten (Fig. 246). 
The pods may also become one-sided in their development. On 
the stems, particularly the upper and softer terminal branches, 
the elongated water-soaked spots end in long sunken black 
stripes (Fig. 247) and the branch either shrivels and dies or is 
broken over. The amount of branch infection varies greatly 
according to the season and the variety. The main stem 
of young plants is also subject to attack and may be killed. 
From the older spots, especially on bolls and stems, there is often 
a yellowish bacterial ooze which dries in the form of yellowish- 
white granules, scales or crusts. 

This disease is widespread in our Southern States and has 
been reported also from other parts of the world. I have 
proved it to occur in Asia (Turkestan) by cultivating my 
organism out of diseased cotton stems received from Tashkent 
(Dr. Schroder's cotton gummosis), and with pure cultures of it 
have reproduced the typical disease in American cotton on both 
leaves and stems in one of our hothouses (1909). I have also 
isolated it from stems of Egyptian cotton grown at Zomba in 



318 



BACTERIAL DISEASES OF PLANTS 





l-v 



Se^ 



'•<* 





Fig. 241. -Angular leaf-spot of cotton showing varying manner of infection 
dependent on age. (See next page.) 



THE ANGULAR LEAF-SPOT OF COTTON: TYPE 



319 



East Africa (Nyassaland), and more recently (1915) from cotton 
grown in Pretoria in S. Africa. In 1915, Bovell and Dash re- 
ported it as serious on late cottons in the Barbados. In 1918 
Reinking reported it from the Philippines. Miss Doidge writes 
me (1919) that she has observed it to be quite common in South 
Africa. 0. F. Cook saw it in China in 1919. Probably the 
disease occurs in all the cotton-growing regions of the world. 




Fig. 242. — Inoculated cotton leaves showing angular spots duetto stomatal 
infection by Bacterium malvacearum. Bacterial suspension sprayed on. Spots 
in second stage, i.e., beginning to shrivel and brown. Time, 6 weeks. Photo- 
graphed April 28, 1915. 3*2 nat. size. 

The extent of damage done by it is unknown. It is worse 
in some localities and some seasons than in others. I regard 
it as a serious disease. 

If any considerable part of the ''shedding" of the young 
bolls is due to it, especially in seasons when shedding is phe- 



1. Result of inoculating cotton leaves when very young. Stomata not then 
open on the parenchyma and attack of Bacterium malvacearum confined chiefly 
to the veins: a, bacteria rubbed by hand on under-surface of right lobe; 6, 
bacteria sprayed on. Photographed April 28, 1915. Time: a, 4 weeks; 6, 6 
weeks. 

2. a, Hand-rubbed (March 25) with Bacterium. maUiac&axwin -on the-under- 
surface of the right lobe when very small and infection confined to the vicinity 
of veins; h, sprayed when older (March 17), i.e., when about half-grown and 
disease confined to the parenchyma. Photographed May 1, 1915. 



320 



BACTERIAL DISEASES OF PLANTS 




Fig. 243. — Cross-section of a cotton leaf inoculated with Bacterium mal- 
vacearum by spraying (uncovered in a hothouse) March 17, 1915. Collected 



THE ANGULAR LEAF-SPOT OF COTTON: TYPE 



321 



nomenally large and interferes with the making of a crop, as now 
seems probable; and especially, if this schizomycete generally 
paves the way for the entrance of the destructive boll fungus, 
Glomerella gossypii (Colletotricum gossypii Southworth), as 
Mr. Orton believes or did believe at one time, then indeed it is a 
very serious enemy of cotton growing. This much is certain, 



^Hlbk.. 










Fig. 244. — Cotton leaf inoculated May 4, 1915, from a windowed colony. Photo- 
graphed June 26, 1915. Time, 53 days. 

the anthracnose fungus and the schizomycete often occur 
together on the bolls in such a way as to indicate that the 
first invasion was bacterial. Further studies are necessary. 
Weather conditions have much to to with the prevalence of 
the disease. 

^ i Cause. — This disease is due to Bacterium malvacearum EFS. 
This organism is a yellow, Gram-negative, non-acid-fast 

and fixed in Carnoy's fluid, April 5. Sectioned from paraffin. Amyl-Gram 
stain. Stomatal region pushed up. X 1000. From a photomicrograph by 
the writer. 

21 



322 BACTERIAL DISEASES OF PLANTS 

(5-day potato cultures stained 10 minutes in Ziehl's carbol 
fuchsin, then washed 1 minute in 70 per cent alcohol containing 
3 per cent hydrochloric acid), non-sporiferous, motile, polar 
flagellate (Fig. 248), feebly liquefying (gelatin and Loffler's 
solidified blood serum), milk-curdling (by a lab ferment), 
non-nitrate-reducing, starch-destroying, sunlight-sensitive (Fig. 
249), dry-air sensitive, frost-sensitive in +15 bouillon (Fig. 250), 
rod-shaped or short catenulate (2 to 4 or more elements) schizo- 
mycete, growing on the surface of +15 agar-poured plates in 
the form of round, thin, flat, smooth, wet-shining, very pale 
yellow colonies which become a deeper color with age but are 
still only pale yellow, i.e., distinctly paler than those of Bacterium 
phaseoli, never deep yellow or orange colored. Well-grown 




Fig. 245. — Green cotton bolls attacked by Bacterium malvacearum. Accidental, 
hothouse infection. Washington, 1904. 

colonies on thin-sown plates measure 1 to 5 millimeters or more 
in diameter. Early in their growth on +15 agar (second or 
third day) the colonies are more or less radiate-mottled and 
this is a good character for separating the parasite from yellow 
cotton saprophytes which often accompany it or follow it 
(Fig. 251 ) . The margin is thin and regular except in old colonies. 



10 spots — pedicel also involved; (2) another boll — photographed end on, showing 
20 or more coalescing spots some of which involve the whole thickness of the 
pericarp, as shown in Fig. 3 at X; (3) same boll as (2) but a side view with the peri- 
carp split open to show the beginnings of the brown stain in the lint — base of the 
pericarp also involved; (4) same as (3) but showing the opposite side of the boll — 
lint at top stained brown. Inoculations of July 7, 1915, made with a "windowed" 
colony of the Arizona organism. The cotton Colletotrichum was not present in the 
hothouse. Photographed by James F. Brewer. (See next page.) 



THE ANGULAR LEAF-SPOT OF COTTON: CAUSE 



323 



1 








# 


•> 




Fig. 246. — Pure-culture inoculations of cotton with Bacterium malvacearum 
showing later stages of boll-spot than Fig. 255: (1) side view of a boll showing 



324 



BACTERIAL DISEASES OF PLANTS 



Often there is a paler zone at the edge of the colony (Figs. 252 and, 
especially, the colony in the upper left corner of Fig, 253) 
or there may be concentric zones as in Bacterium phaseoli. Oc- 
casionally the mottling of surface colonies on agar is so conspicu- 
ous as to suggest an intruder (Fig. 254), but inoculations with sub- 
cultures from such a colony produced thousands of typical spots 
on cotton leaves (Fig. 244) and also infected the bolls typically 




Fig. 247. — Rather woody cotton stems attacked by Bacterium malvacearum 
as the result of needle prick inoculations (Nos. 103-111). Sept. 19, 1905. Photo- 
graphed Oct. 1, 1905 but with insufficient contrast. The "black arm" of the 
planters. 

(Fig. 255). Moreover, after a few days, such strikingly mottled 
colonies fill up their thin places, as maybe seen by comparing Figs, 
253 and 256. To these curious forms which suggest rose- win- 
dows we have given the name 'Svindowed" colonies. 

The colonies in + 10 peptone beef gelatin plates are also 
characteristic. They are yellow and circular, A feeble pit of 
liquefaction is produced and the colony sends into the depths 



THE ANGULAR LEAF-SPOT OF COTTON: CAUSE 325 

of the softened gelatin (Arizona organism) numerous spatulate 
finger-like projections (Fig. 257). Gelatin stab cultures are 
liquefied very slowly. In tube cultures 83 days old, one-half 
to two-thirds only of the gelatin was liquefied. There was no 
decided rim or pellicle, but a copious yellow precipitate, the 
fluid gelatin remaining clear and unstained. In another year 
(1915) tubes of +10 peptone beef gelatin inoculated with the 
Arizona organism by needle stabs were only one-fourth liquefied 
after 30 days at 16° to 19°C. although there was a prompt and 



^ 


'• ^. 




• 




* 


'' ^ 






^ 




/»' 


■■-. ,- 




1 


i - 


p 


^ 


/ 






«* ' 


^ 


/ 



Fig. 248. — Flagellate rods of Bacterium malvacearum cultivated from an 
angular leaf spot. Arizona cotton. Stained by van Ermengem's silver method. 
X 1000. 

good growth. At the end of 60 days (same temperature) less 
than one-half the gelatin was liquefied. 

On Loffler's solidified blood serum, there is a copious growth 
(paler yellow than that of Bacterium phaseoli) with slow lique- 
faction. At end of 15 days in tubes containing Bacterium 
phaseoli all the substratum had changed color (darkened) and 
most of it had liquefied ; whereas in tubes containing Bacterium 
malvacearum there was very little change in color (the bulk 
white) and not one-twentieth part had liquefied. At the end of 
30 days the difference in color and amount of liquefaction was 



326 



BACTERIAL DISEASES OF PLANTS 



still marked. Experiment repeated in 1919 with the same dis- 
tinct differences (Fig. 258). 

On steamed potato cylinders standing in water there is at 
first a thin, pale yellow, wet-shining growth, which soon becomes 
copious and entirely fills the fluid, making it solid. The color 




Fig. 249. — Agar-poured plates of Bacterium ■malvacearum showing appearance 
5 days after exposure (on ice) of the right half to bright sunlight for 2 minutes: 
A. Bacteria isolated from Turkestan cotton in 1909; B. Bacteria isolated from 
Arizona cotton in 1914. 



is then Naples yellow to wax-yellow (Ri), becoming brownish 
with age. The potato grays more or less, and the starch is con- 
sumed (see No. II and No. VIII). Experiment repeated in 
1915 using the Arizona organism with the same result. The 



THE ANGULAR LEAF-SPOT' OF COTTON: CAUSE 327 

growth was copious, smooth, gUstening and- the color after a 
month w^as mostly between Ridgway's light cadmium and his 
empire yellow^ (R2), the dark stain ranging from his orange 
citrine in the slime to his mouse gray in the potato, and all but 
an insignificant residue of the starch was consumed. 

In peptone-beef bouillon there is a moderate and persistent 
clouding, the best growth at first in unshaken tubes being at the 
top. There is a pale yellow rim and a moderate maize yellow 
precipitate. There are some pseudozoogloeae. 

It grows moderatel}'^ in Uschinsky's solution with a pale rim 
and considerable flocculence. It grows feebly or not at all in 
Cohn's solution. 

The thermal death-point lies between 50° and 51°C. but is 
never 50°C. (tests of 1919 in -f-15 peptone beef bouillon). 
The maximum temperature lies between 36° and 38°C. — repeated 
in 1919 it grew at 35° and not at 37° and after 14 days at 37° 
the tubes did not cloud at room temperature (6 da3^s) . It grows 
slowly at 10°C. There w^as no growth in -f-lo peptone-beef 
bouillon in 6 weeks at 8°C. Repeated in 1919 faint clouding 
after 10 days at 8.5° to 10.5°C.; the tubes at 6° to 8°C. remained 
clear for 21 days (as long as tested). 

Tubes of plain milk inoculated in 1919 gave after 43 days 
the result shown in Fig. 259. At this time there were no tyrosin 
crystals. The whey, sterilized at 54°C., or kept in the 
thermostat at 38-44°C., caused prompt precipitation of the curd 
when added to sterile milk and subsequent transfers from these 
tubes to suitable media showed them to be free from bacteria. 

In litmus milk the litmus is blued and the casein is thrown 
down slowly. The litmus may be reduced, but is not reddened, 
so that the existence of a lab ferment is inferred. The precipi- 
tated curd is not promptly digested but subsequently most of 
it disappears (2 months), and tyrosin crystals not visible at 
first (30 to 40 days) are then abundant (Fig. 260). Compare 
with No. II (Fig. 93). 

This organism is extremely sensitive to light, even 2-minute 
exposures (on ice) being enough to clear the sunlit one-half of 
thickly sown agar-poured plates, and even 1 -minute exposures 



328 



BACTEEIAL DISEASES OF PLANTS 




Fig. 250. — Agar poured plates showing effect of freezing on Bacterium nialva- 
cearum: Arizona organism. B. Check plate. A. Same quantity of the culture, 
sowed after freezing for 1 hour in +15 peptone-beef bouillon. 



THE ANGULAR LEAF-SPOT OF COTTON: CAUSE 



329 



destroyed the greater number (Turkestan, organism; Arizona 
organism). Contrast with No. VIII. 

In various ways this organism resembles Bacterium campestre, 
Bacterium phaseoli and Bacterium citri but I did not succeed in 
cross-inoculating it to cabbages, to beans or to oranges. Fur- 




FiG. 251. — Part of an agar-poured plate of Bacterium malvacearum enlarged 
to show fugitive motoling of the surface colo nies. From one of the leaf spots shown 
in Fig. 240. Time, third day. Temperature 23°C. Plating of March 20, 1915. 
X 14. 



ther comparisons should be made not only with Bacterium cam- 
pestre, Bacteriu77i citri, and Bacterium phaseoli, but also with 
Bacterium pruni, Bacterium juglandis, and Bacterium translucens, 
all of which are closely related. Indeed, some of these names are 
perhaps synonyms, but this can be settled only by many cross- 
inoculations and much further study. 



330 



BACTERIAL DISEASES OF PLANTS 



Technic. — Sometimes there are difficulties in the way of 
isolating this organism, owing to the occurrence on the cotton 
plant of yellow saprophytes which somewhat resemble it. These 
are often found in the spots or on the surface and are perplexing 




Fig. 252. — Bacterium malvacearum: part of an agar-poured plate, enlarged 
to show fugitive mottling of the surface colonies. From one of the leaf spots 
shown in Fig. 240. Time, third day. Temperature 23°C. Plating of March 
22, 1915. X 14. 

on the agar-poured plates. For this reason great care should 
be taken to make the isolations from clean leaves, stems and 
bolls in early stages of the disease, selecting typical-looking spots 
which have not yet passed beyond the translucent watery- 



THE ANGULAR LEAF-SPOT OF COTTON: TECHNIC 331 

looking stage of the disease. Do any of . the accompanying 
saprophytes favor the growth of the parasite? 

Only yellow colonies should be considered. jNIoreover all 
yellow ones that have a wrinkled surface, all that reduce nitrates, 
redden litmus milk or fail to throw down the casein must be re- 
jected on the start. Potato cylinders also should be used as a 
means of separation, all orange-colored and slow-growing, non- 
starch-consuming organisms being rejected. On agar-poured 
plates besides the white colonies and wrinkled yellow ones, cir- 
cular pale yellow colonies of two or more types may appear. 
Those having a smooth surface and a mottled interior, i.e., light 
and dark aggregations, are likely to be the right organism, 
especially if they give a copious pale yellow growth on potato 
and precipitate casein from litmus milk without development of 
an acid. Those pale yellow surface colonies having from the 
beginning (2d day, 3d day) a uniform inner structure and yield- 
ing a scanty creamy growth on the potato cylinders should be 
rejected. Fortunately, young rapidly growing cotton plants 
are quite susceptible and sub-cultures from the various colonies 
may be tested out quickly, by means of stem and leaf inocula- 
tions, care being taken, if the sprayed plants have been shut up 
over-long in cages, that suffocation spots are not mistaken for 
bacterial spots, since the former may occur. Sections (Fig. 261) 
will quickly show whether or not the spots contain bacteria in 
numbers. 

Cotton for the inoculations should be planted in a warm 
hothouse six weeks to two months before it is needed. Many 
varieties are subject to the disease. I have had good success 
with inoculations on Rivers, Sunflower, Durango and Columbia. 
The disease is readily induced on young rapidly growing stems 
and bolls by delicate needle punctures introducing the parasite, 
and se^'eral times I have produced it on leaves simply by gently 
rubbing my infected fingers over their undersurface (Fig. 241aa). 
Generally, however, for the leaf infections, the spraying of water 
suspensions of young agar-streak cultures upon plants shut up 
48 hours in spraying cages will prove most satisfactory. Cages 
are not necessary, however. I have not seen the leaf spots due to 
stomatal infections appear earlier than toward the end of the 



332 



BACTERIAL DISEASES OF PLANTS 



second week. As in many other diseases (see No. V), once a 
few plants are infected, the gardener's hose is a ready means of 
distribution to others (Fig. 255), just as a driving rain (Faul- 
wetter) is in the open field. 




Fig. 253. — Photograph of three surface colonies of Bacterium malvacearum, 
the middle and right of which were windowed conspicuously on the fourth day 
(see Fig. 256). These colonies also show the pale rim. Several buried colonies 
are also visible. X 7. The lines are pencil marks on the bottom of the plate 
indicating to the photographer the colonies to be photographed. 

Before trying isolations from leaf-spots we always plunge 
the leaf momentarily into 1 : 1000 mercuric chlorid water to 




Fig. 254. — Bacterium malvacearum. Agar-poured plate of March 22, 1915. 
Photographed March 25. Typical transient mottling. From the Arizona 
cotton. X 14. 

discourage surface organisms, but the exposure should never be 
for more than 20 to 60 seconds. The piece may then be thrown 
into*'sterile water and rinsed {always briefly, lest the [poison 



THE ANGULAR LEAF-SPOT OF COTTON: TECHNIC 



333 



be removed and the surface bacteria restored to a growing con- 
dition), then the spot is crushed thoroughly in bouillon and, 
after a half-hour's delay with more or less shaking to diffuse 
the bacteria, the plates may be poured. 

Determine 

For THE ORGANISM. Morphology. — Size in microns, form 
(stain with Ziehl's carbol fuchsin or amyl Gram), aggregation 
of elements, motility (margin of a hanging drop), number and 



/f 







Fig. 255. — Accidental inoculation of Bacterium malvacearum on cotton 
bolls by spraying, July 7, 1915. Photographed August 2. These are secondary 
infections by means of the gardener's hose and are approximately 2 and 3 weeks 
old. 

location of fiagella (van Ermengem's silver-nitrate stain), 
absence of endospores (heat, stains), presence or absence of 
capsule (Ribbert's capsule stain), occurrence of involution forms, 
reaction to Gram's stain, acid-fast stain. 

Cultural Characters. — On nutrient agar, on gelatin ( + 10), 
on Lofiler's solidified blood serum, on steamed potato, in 



334 



BACTERIAL DISEASES OF PLANTS 




Fig. 256. — Buried and surface (rose-windowed) colonies of Bacterium mal- 
vacearum on an agar-poured plate. Photographed March 24, 1915. X 14. 
For appearance of these colonies two days later see Fig. 253. 



LEAF-SPOT OF COTTON: CULTURAL CHARACTERS 335 

peptone bouillon, nitrate bouillon, Cohn's solution, Uschinsky's 
solution, Fermi's solution, litmus milk, peptone water in fer- 
mentation tubes with various pure sugars and alcohols. Com- 
pare with No. II and No. VIII. 

Non-nutritional Environment. — What is the optimum tem- 
perature for growth? the maximum? the minimum? Can 
you get any growth at 1°C. or at 37°C.? Compare critically 
with No. VIII. Effect of sunlight (try first a 2-minute exposure 
on a bright day) ? Effect of dry air (use 2-day peptone-bouillon 
cultures spread on thin sterile cover-glasses — begin at G hours; 




Fig. 257. — Spatulate, finger-like out-growths of a gelatin colony of Bacterium 

malvacearum. 

thereafter drop some of the infected covers into suitable bouillon 
every 6 hours — Keep covers in a dark place) . Effect of freezing ? 
of weak acids? of sodium hydrate? of chloroform in bouillon? 
Maximum toleration of sodium chlorid in bouillon? Length of 
time organism will remain alive inside of leaf-spots, or boll- 
spots? or in cotton stems? It is a very considerable period. 
For the disease. Signs. — How soon does the disease 
appear on the inoculated (sprayed or smeared) leaves? How 
soon on punctured bolls or stems? How soon do the spots 
change from translucent to brow^n? Are leaves or bolls com- 
monly thrown off as a result of infection ? Inoculate very young 



336 



BACTERIAL DISEASES OF PLANTS 



bolls and watch. Determine the effect of the disease on young 
seedlings, inoculating cotyledons and hypocotyl by means of 
needle pricks. Describe the disease, but not out of this book. 
Histology. — Embed, section and stain young leaf-spots, 
18th day and earlier. Can you find distinct evidence of stoma- 




FiG. 258. — Tubes of Lofiier's solidified blood serum inoculated from bouillon 
with Bacterium malvacearum (Arizona organism) and with Bacterium phaseoli 
(Idaho organism) for comparison; Nos. 1 and 2 streaked April 12; 3 to 6 streaked 
April 21. Photographed May 3, 1919. No. 1, liquefied; Nos. 3 and 4, partly 
liquefied; Nos. 2, 5 and 6 not liquefied. No. 2 is drying out on right side. 

tal infection? Stain with carbol fuchsin or amyl Gram. In 
the same way study early stages of the disease on stems and bolls. 
Does a brown stain accompany this disease? 

Variability. — Does this disease ever kill the plant? Ever 
render it unprofitable? Attack some varieties to the ex- 
clusion of others? Appear to be much worse some seasons 



THE ANGULAR LEAF-SPOT OF COTTON: VARIABILITY 



337 



than others? Is it worse on lowlands than on uplands? Is it 
modified in any way by methods of culture? Are fungi as- 
sociated with it? Try mixed infections. Study the effect of 
the disease on seedlings in the hothouse and in the field. 

Transmission. — This disease is probably transmitted on the 
seed, but we have as yet no experimental proof of this. Can 
you help to clear up the situation? Once established in the 
field, is. it ever spread except by the wind during rain storms? 




Fig. 259. — A. Baderiiun vialvaccarinn, Col. 4 (Arizona isolation), after 43 
days in milk at 25°C. Casein precipitated by a lab ferment. B. Check tube. 
Milk dried out about one-fourth. 



Read Faulwetter's paper and look tor wind-driven paths of 
infection. Kellerman reports similar paths oi citrus canker. 

Does the disease occur on weeds, or on any other cultivated 
plants? Try inoculations on okra, abutilon and hollyhock by 
spraying. 



338 



BACTERIAL DISEASES OF PLANTS 




Fig. 260. — Tyrosin crystals from an old litmus-milk culture of Bacterium 
malvacearum. Arizona cotton, culture of March 20, 1915. Compare with Fig. 93. 
Photographed June 3. X 5. 




Fig. 261. — Section through an early stage of an angular spot on a cotton leaf, 
showing the bacteria (at X) extruding through a stoma in the upper epidermis. 
Dillon, S. C, 1900. 



THE ANGULAR LEAF-SPOT OF COTTON: LITERATURE 339 
LITERATURE 

The writer named the organism Pseudomonas malvacearum 
in 1901 (Bull. 28, Div. Veg. Phys. and Path., U. S. Dept. of 
Agriculture, p. 153) but this is the first time he has carefully 
described it. (This chapter was written in 1915 previous to the 
appearance of Faulwetter's papers, which see.) There are 
references to the disease in various Experiment Station 
publications. 

Read especially ''The Rots of the Cotton Boll" by C. W. 
Edgerton (La. Agr. Exp. Sta. Bull. No. 137, Baton Rouge, Dec, 
1912) and "Dissemination of Angular Leaf Spot of Cotton," 
by R. C. Faulwetter (Jour. Agr. Research, March 19, 1917, pp. 
457-475); also by the same author "Physiology of Bacterium 
malvacearum Smith" (27 Ann. Report, S. C. Exp. Sta., 1917). 

Consult also '' Bacteria in Relation to Plant Diseases," 
Vol. I (1905), Figs. 80 and 118, and Vol. II (1911), Fig. 18 and 
statements in text (See Index). 



XL THE MULBERRY BLIGHT 

Type. — This is a blight in many respects resembling fire 
blight of the pear (No. XII) but slower in its action and with 
a correspondingly greater tendency to spotting and distortion 
of the leaves (Figs. 262, 263), in which it resembles the walnut 
blight, and the leaf-spot of beans (No. VIII). Dark, sunken, 
longitudinal stripes preceded and bordered by a translucent 
area also appear on the young shoots. These sunken spots 
often show a whitish or yellowish bacterial ooze, drying glossy. 
If the surface is dry this ooze often exudes from the lenticels 
in the form of cirri (Fig. 264). In such young shoots both 
wood and bark are invaded by the bacteria, and the end of the 
disease is either shriveling and death of the shoots (Figs. 265 to 
267), or a curved one-sided growth. As in pear blight, we have 
to do, primarily, with a bark disease (Figs. 268, 269). The 
disease usually begins in the shoots of the season (F^g. 270), 
but the blight frequently extends beyond these, especially into 
shoots of the previous year. On the older parts the disease 
occurs in the form of cankerous patches. As in fire-blight 
of the pear, the tendencA^ of the bacteria to ooze to the surface 
is strong. 

On the leaves there are numerous coalescing and slowly 
enlarging spots which are water-soaked in appearance at first, 
then brown or black, the surrounding tissues becoming yellow. 
The bacteria occupy the intercellular spaces and form cavities 
(Figs. 271, 2725). On the midrib and veins dark sunken spots 
appear, similar to those on the shoots. Leaves attacked early 
in their development become variously distorted (see also No. 
VIII). 

So far as known, mulberry trees are seldom or never killed 

340 



THE MULBERRY BLIGHT: TYPE 



341 



outright by this disease (except sometimes nursery stock), but 
the trees often become ragged, stunted, and unprofitable. As 
in fire-blight on the apple, the crown of the tree may be spotted 
all over with conspicuous dead twigs bearing brow^n leaves. 




Fig. 262. — A mulberry shoot 9 days after inoculation with Bacterium mori. 
Shows dark sunken places on the stem (where pricked); younger leaves shriveling; 
older leaves spotted and distorted. Dept. of Agric. hothouse, Jan. 13, 1909. 

The disease prevails on various kinds of mulberry, including 
the Russian. It cannot be produced by inoculating with the 
pear-blight organism. 

Its exact geographical distribution is not known. It occurs 
in various parts of the United States (East, South, and West), 



342 BACTERIAL DISEASES OF PLANTS 

and I have seen it in France. It is very common also in South 
Africa (Miss Ethel M. Doidge), Prof. Gentaro Yamada, who 
has seen Arnaud's experiments in Paris, tells me that the dis- 
ease is common on the mulberry in Japan. It probably occurs 
also in Italy, Russia, and Australia. The doubt about Italy, 
where for a long time the same or a similar disease has been 
known, is one concerning exact identity. Several Italian path- 
ologists have studied the Italian disease but I am inclined to 
doubt the value of their bacteriological findings; at any rate they 
have ascribed the disease to Bacillus cuhonianus Macchiatti, 




Fig. 263. — Distortion of South African mulberry leaves due to Bacterium mori. 

After Ethel M. Doidge. 

a liquefying, yellow schizomycete. Yellow bacteria are very 
common on mulberry trees and are often present in the dis- 
eased areas, but are not the cause of the disease now under 
consideration. 

Cause. — This disease is due to Bacterium mori Boyer and 
Lambert emend. EFS. 

Here some introductory remarks are necessary on the prob- 
lem of what to do with an old name when it has been given with- 
out a proper characterization. This problem is often a difficult 
one. Always, if possible, I think the first name should be re- 
tained by the pathologist. It cannot be, however, especially 



THE MULBERRY BLIGHT: CAUSE 



343 



in the absence of successful inoculations, if the author has tied 
it to definite characters incompatible with parasitism, and the 
organism in question is definitely parasitic. But sometimes, as in 
this instance, contradictory statements are made. In such cases I 
think we may retain the name and reject the contradictions pro- 




FiG. 264. — Diseased stems of mulberry showing extrusion of bacterial cirri 
{Bacterium mori) through lenticels. Inoculated January 4, 1909. Photo- 
graphed January 11. 

vided there is a definite history of pathogenicity connected 
with it. For these reasons I discard Bacterium oleae Archangeli 
for which there is no history of pathogenicity and no proper 
characterization (see No. XIII), and retain Bacterium mori 
Boyer and Lambert where there is a correct account of the 



44 BACTERIAL DISEASES OF PLANTS 

disease and a definite history of successful inoculations but a 
very imperfect description of the parasite, one statement in 
which is erroneous. 

Petri gives Bacterium mori B. and L. and Bacillus cuhonianus 
Macchiatti as synonyms of Ascohacterium luteum Babes. The 
latter may be, since it is described as yellow, capsulate and lique- 
fying (sporiferous, however, according to Macchiatti) but the 
former cannot be since Boyer and Lambert obtained with it a 
disease due to a white organism. The mulberry blight of the 
United States, of France, and of South Africa, is identical and 
is due to a non-liquefying white schizomycete, as proved inde- 




FiG. 265. — South African mulberry twigs killed by Bacterium mori. After 

Ethel M. Doidge. 

pendently in the United States by Smith, Rorer, and O'Gara, 
in France by Arnaud and by Smith, and in South Africa by 
Ethel M. Doidge. My observations of the signs and lesions of 
the disease made both in the United States and in France cor- 
respond closely to Boyer and Lambert's description of their 
disease. They state that with the parasite taken from blighting 
stems of the mulberry they produced the characteristic disease 
on the parenchyma and in the veins of the leaf, but their de- 
scription of this parasite is limited to the bare statement that 
"Isolated and cultivated on the surface of artificial solid media, 



THE MULBERRY BLIGHT: CAUSE 



345 



Bacterium mori gives hemispherical colonies which from h3'aline- 
white [correct statement] pass over into yellow [incorrect state- 
ment as a whole, although on some media, e.g., Loffler' solidi- 
fied blood serum, it is somewhat yellowish]." They promised a 
farther report but made none. We may assume that they had 
mixed cultures on the start or soon after, viz., the parasite and 







Fig. 266. — Branch of Morns nigra (black mulberry) attacked by Bacterium 
mori. Collected at Montpellier, France, in July, 1913. Section of axis at right. 
After Arnaud and Secretain. 



some yellow saprophyte, and that no further report was made 
because having turned their attention to the wrong organism 
they could not get any more infections and became confused. 
Non-parasitic yellow organisms are so common in the mulberry 
lesions that following Macchiatti's misleading statements it 



346 



BACTERIAL DISEASES OF PLANTS 



would be very easy to make this mistake in the early stages of 
the investigation. I made it myself. 

]\Iacchiatti's name Bacillus cubonianus, is earlier by one year 
than Boyer and Lambert's name, but unfortunately he made no 
inoculations and ascribed to his organism characters definitely 
excluding it from any role in the causation of the mulberry dis- 
ease here described, i.e., formation of endospores, presence of a 
capsule, yellow color on media, liquefaction of gelatin, etc. His 
name, therefore, may be reserved for consideration in connec- 
tion with the Itahan disease, in case there should be one different 
from the French disease. According to ]Macchiatti the behavior 




Fig. 267. — Branch of Morus alba (white mulberry) attacked by Bacterium mori. 
September, 1913. After Arnaud and Seer etain. 

of his Bacillus cubonianus is very typical on potato where from 
the beginning there is a rapid growth with the formation of large, 
slightly raised, sinuous-margined colonies having a j-ellow color, 
which becomes ever more intense with age. 

Boyer and Lambert's Bacterium mori is therefore the earliest 
available name for the cause of this disease. We must either 
accept that or devise some entirely new name. It is almost a 
nomen nudum, but not quite, since it was isolated from diseased 
(blighting) mulberries and has a definite if inconsiderable history 
of pathogenicity attached to it, as the result of successful inocu- 
lations. In my first paper (1910), therefore, I felt entirely free 
to retain their name and to attach to it a description drawn from 



THE MULBERRY BLIGHT:" CAUSE 



347 



the American species, believing the French organism could not 
be different from our own. In this belief I was entirely right, 
as subsequent observations and experiments have proved (1913). 
With these introductory comments we may proceed to a 
statement of the characters of Bacterium mori B. and L., drawn 
entirely from studies made in mx laboratory, using cultures de- 
rived both from American and French sources. 




Fig. 268. — Cross-section of young stem of mulberry showing the inner cortex 
honeycombed by cavities due to Bacterium mori. A natural infection from 
Arkansas. 1908. Medium magnification. 

Bacterium mori B. and L. emend. EFS., is a white, non-viscid 
or slightly viscid (potato), slow-growing, non-sporiferous, non- 
capsulate, actively motile (1-7 polar flagella, usually 1-4), 
Gram-negative, non-gas-forming, strongly aerobic, non-lique- 
fying (gelatin and Lo filer's solidified blood serum), non-nitrate- 
reducing, acid-sensitive (Cohn's solution), sunlight-sensitive, 



348 BACTERIAL DISEASES OF PLANTS 

heat-sensitive, chloroform-tolerant in +15 peptone beef bouillon 
(grows unrestrictedly), dry air-tolerant (30 to 50 days or more), 
sodium chlorid-tolerant (to 6 or 7 per cent in +15 peptone 
beef bouillon) non-coagulating (milk), casein translucing (milk 
is cleared as by No. IV), rod-shaped, paired, clumped or 
catenulate schizomycete, forming on the surface of +15 nutrient 
agar-poured plates slow-growing, translucent, white, circular, 
smooth, flat colonies, entire at first but becoming undulate- 
margined after some days, and having for a time, like many other 
colonies, a reticulate or striate internal structure. 

•Growth on young agar-streaks moderate, white, odorless, 
translucent, spreading, flat, dull to shining, smooth, becoming 
finely granular; medium not stained. Contrast with No. IV. 

Colonies on +10 beef-bouillon peptone gelatin slow-grow- 
ing, white, flat, circular to irregular with lobate erose margins 
(compare with XIII). Growth in gelatin stabs fihform, best 
at top; no stain, no liquefaction. 

Growth on Lofiier's solidified blood serum, scanty to 
moderate, yellowish white. No liquefaction — not even after 
many days. 

Growth on steamed potato moderate, spreading, flat, glisten- 
ing, smooth, white to dirty-white (medium grayed, never 
blackened), action on the starch slight. 

Growth in +15 peptonized beef broth always best at the 
top where a pellicle develops which fragments easily on shaking 
and sinks, forming a flocculent, turbid, odorless fluid (clear 
after 3 months). 

Milk is not coagulated but becomes translucent (by some 
change in the casein); it is then strongly alkaline and not 
viscid. After 3 months and considerable evaporation the fluid 
is gelatinous and somewhat brownish (near Saccardo's ochro- 
leucous). Purple litmus milk becomes blue; there is never 
any acid reaction (compare with No. IV). 

In Cohn's solution no growth or only a very slight growth 
(contrast with No. XIII). 

In Uschinsky's solution a copious growth and a heavy 
fragile pellicle, sinking readily. Fluid bluish-fluoresdent (5th to 
10th day), not viscid. Repeated in 1919 : uniforml}^ well clouded 



THE MULBERRY BLIGHT: CAUSE 



349 



on 4th day; best growth in top on 6th day when it was bluish- 
fluorescent; many pseudozoogloeae; no distinct pelUcle; after 6 
weeks, greenish-fluorescent, very copious white precipitate and 
still heavily clouded (contrast with XIII); organism motile; no 
filaments observed. 

In peptone water in fermentation tubes no clouding of the 
closed end with dextrose, saccharose, lactose, maltose, glycerin 
or mannit. Contrast with No. I. 

Indol production is absent or feeble. Compare with No. VI. 



^s-- 







- 7 -75^- 



*rf« 






Fig. 269. — Bacteria of mulberry blight: a. Bacterial cavity in bark of the 
young mulberry shoot shown in Fig. 268; most of the bacteria were washed away 
in making the preparation. Remnants of tissue on the periphery. Over-stained. 
X 1000. 

b. Center of small cavity in bark of a mulberry shoot inoculated on grounds 
of the V. S. Department of Agriculture in 1910. X 800 circa. 



Grows from 1°C. to 35°C. but remains alive in bouillon for 
only a short time at the latter temperature. Thermal death- 
point about 51.5°C. 

Grew twice in +15 peptone bouillon containing 7 per cent 
sodium chlorid but would not tolerate 9 per cent . (Compare with 
No. VI and contrast with No. I.) 

Pseudozoogloeae and involution forms occur. 

On thin-sown agar plates all bacteria on the exposed side 
were killed by 36 minutes exposure to sunlight, bottom up on 
ice; 26 minutes killed nearly all. 



350 



BACTERIAL DISEASES OF PLANTS 





Fig. 270. — Leaves and shoots of mulberry attacked by Bacterium mori. From 
Georgia. May, 1905. J2 nat. size. 



THE MULBERRY BLIGHT: CAUSE 351 

Bacterium mori does not lose virulence readily. Cultures 
of the Georgia organism (Berckmans I and II) carried along 
on culture media in my laboratory for 12 years (1908-1920) 
were stiU infectious. Contrast with Xos. IV and XIV. 

Technic. — At first the writer isolated the wrong organism, 
owing to misplaced confidence in European statements re- 
specting its color. All the various types of yellow colonies 
appearing on the agar-poured plates were subcultured and 
inoculated into growing mulberry shoots, but to no purpose. 
The yeUow bacteria would not produce the disease. In this 
way a whole summer was wasted. The following year, however, 
no difficulty was experienced in plating out an actively patho- 
genic white schizomycete. 

For the isolations the student should select, from stem or 
leaf, clean parts recently diseased and swarming with the 
bacteria as determined by a microscopic examination. The 
surface of the part selected should be flamed Ughtly, if stem; 
or plunged for a minute or two into 1 : 1000 mercuric chlorid 
water, if part of a leaf, and then rinsed lightly in sterile water 
(long soaking in water removes this poison and may resuscitate 
the surface bacteria which one desires to kill or to keep dormant 
for a day or two; on the contrary a Uttle of the surface poison 
carried over into the bouillon does no harm). If it is a leaf-spot, 
the piece may now be thrown into a tube of bouillon and crushed 
with a sterile glass rod. If stem, the diseased part should be 
scraped out with a cold sterile instrument and thrown into 
bouillon. In either case the tissue should be allowed to soak 
for an hour before the dilutions are made and the plates poured. 
Plates should be sown both from the original tube and from the 
dilutions, seeding some heavily and others lightly. All yellow 
colonies appearing on the plates should be rejected unless it is 
desired to study also the saprophj-tic followers of the parasite. 
For this reason it is best to keep the agar-poured plates under 
observation several days before making transfers, since the 
yellow colonies are frequently quite pale at first and if picked 
off when small or from plates sown too thickly might be mis- 
taken for white colonies. 

For inoculation, select young growing leaves and shoots, 



352 



BACTERIAL DISEASES OF PLANTS 




Fig 271 —South African mulberry blight due to Bacterium niori: section 
through a water-soaked spot on a leaf of the common mulberry which was fixed 
5 days after inoculation, showing lower epidermis lifted, a large bacterial cavity 
and penetration of the intercellular spaces of the mesophyll. After Ethel M. 
Doidge. 



THE MULBERRY BLIGHT: TECHNIC 



353 




tnj) u. 




Fig. 272.— a. Flagellate rods of Bacterium mori. X 1500. B. A detail of the 
mesophyll from Fig. 271. Both after Ethel M. Doidge. 
23 



354 BACTERIAL DISEASES OF PLANTS 

pricking each several times with a deUcate needle infected from 
young potato or agar-streak cultures. Leaves should be inocu- 
lated in the midrib or main veins as well as in the parenchyma. 
Small trees suitable for inoculation may be obtained from 
nurserymen and planted in the autumn for late spring inocu- 
lations out of door, or in the hothouse for inoculations in late 
winter and early spring, whenever shoots are pushing. 

Determine 

For the organism. Morphology. — Size in microns (diam- 
eter is more important than length, but that also should be 
recorded, examining both young and old cultures on various 
media). Conditions under which chains are formed. Ditto 
pseudozoogloeae. Ditto involution forms (use peptonized beef 
broth with 6 per cent sodium chlorid). Motility (on margin 
of a hanging drop). Number and attachment of flagella (use 
Pitfield's stain). Ethel M. Doidge in South Africa, finds 
1 to 4 polar flagella (Fig. 272 A). In my first studies of the 
Georgia organism I saw only 1 to 2 polar flagella, but slides 
stained for me in 1915 by Mary Katherine Bryan, using the 
French organism and van Ermengem's silver-nitrate stain, show 
1 to 7 polar flagella on backgrounds very free from detritus 
or artefact lines (Fig. 273). Absence of endospores (heat, spore 
stains), existence or non-existence of a capsule (Ribbert's dahlia 
stain) . Reaction to various other stains including Gram's stain, 
and acid-fast stain. 

Cultural Characters. — Appearance in poured plates of +15 
peptone beef agar (Figs. 274, 275), streaks and stabs. Ditto +10 
beef peptone gelatin. Growth on Lo filer's solidified blood serum 
(the streak should be flat, white, smooth and glistening). Is 
there any liquefaction of gelatin or solidified blood serum? 

Behavior on potato cylinders. Test, when growth has ceased, 
for destruction of starch. 

Appearance in +15 peptonized beef bouillon. Action, if 
any, on potassium nitrate in peptonized beef bouillon. Beha- 
vior in milk and litmus milk (watch very carefully the early stages 
for absence of coagulation and acidity and the late stages for 



THE MULBERRY BLIGHT: CULTURAL CHARACTERS 355 

clearing, keeping uninoculated tubes for comparison; after 10 
weeks examine under the microscope for general appearance, 
comparing with one of the check tubes). Growth in Cohn's 
solution (there should be none or very little). Growth in 
Usehinsky's solution (which should be copious). 

Behavior in fermentation tubes in peptone water (Why not 
in beef bouillon?) containing various sugars and alcohols (which, 
of course, must be pure). Any growth in the closed end? 
Any acids formed in the open end? 





' t. 




"^ 


> 


^ 



Fig. 273. — Flagellate rods of Bacterium mori. Stained by van Ermengem's 
method from a 2-day agar streak. James Birch Rorer's isolation. X 1000. 

Determine production of indol using Bacillus coli for com- 
parison and testing at end of 10 days and 20 days (heat the 
tubes in a water bath at 80°C. for five minutes, if necessary). 

Non-nutritional Environment. — Minimum temperature for 
growth (try first at 2°C.)? Maximum temperature {irv first 
at 34°C.)? Can you obtain any growth at 1°C. or at 37°C.? 
Optimum temperature (try first in peptone bouillon at 27°, 30°, 
and 33°C., inoculating copiously from bouillon and examining 
every 3 hours)? Is the organism sensitive to dry air (to have 
all the bacteria freely exposed, use young bouillon cultures) ? 
Effect of sunhght (expose on ice for 10, 20, 30, and 40 minutes 
in thin-sown agar-poured plates, preferably after a storm, i.e., 



356 



BACTEEIAL DISEASES OF PLANTS 



when the sky is free from clouds, dust or haze, keeping one-half 
of the plate covered). 

Effect of sodium chlorid in +15 peptonized beef bouillon 
(begin with 5 per cent and increase to 10 per cent, comparing 
on the one hand with Bacillus carotovorus and Bacillus coli and 
on the other with Nos. I and V). 



a 









Fig. 274. — Surface and buried colonies of Bacterium mori on agar poured 
plates, a. Photographed by direct transmitted light, b. The same by oblique 
transmitted light, showing the internal markings. These colonies are smooth 
on the surface and glistening white by reflected light but pale brownish by direct 
transmitted light. X is a thin colony on the bottom of the plate. Time, 5 daj'S. 
Photographed February 10, 1919. X 10. 

Growth in test tubes of unshaken bouillon over chloroform 
(Compare with No. II). 

Nothing is known respecting the action of germicides. 
Can't you make some tests? 

For the disease. Signs. — Period of incubation in young 
stems and leaves. Time from appearance of water-soaked 



THE MULBERRY- blight: SIGNS 357 

spots to the blackening of leaves and shoots. How far down 
the stems in advance of external signs can you trace an internal 
stain? In what tissues? Effect of the bacteria on the tree 
as a whole. 

Describe the disease. Make photographs or drawings. 

Histology. — Cut, freehand, various cross-sections and longi- 
tudinal sections of affected stems, and examine at once in water. 
Fix, embed, section and stain suitable pieces of stem and leaf 
in early stages of the disease to show the bacterial invasion. 
Make permanent preparations. Study ooze of the bacteria 
through lenticels; section young stems some inches below 
external signs of the blight for presence of tyloses in the affected 




Fig. 275. — Agar surface colony of Bacterium mori showing internal markings 
when viewed by transmitted oblique light. Colony smooth on the surface. 
X 23. 

vessels. Can you find any in normal shoots of this age? Com- 
pare with tomato and potato stems attacked by No. IV or VI, 
which also show tyloses. What substances cause their produc- 
tion (see Figs. 357, 358 and 359 for tyloses induced by purely 
chemical means) ? How many inches can you trace the bacteria 
downward in the vascular system below the lowermost external 
indications of the disease? Can the tyloses be traced farther 
than the bacteria? What stem- tissues are specially involved? 
For answer to this question examine both the soft terminal part 



358 BACTERIAL DISEASES OF PLANTS 

of blighting shoots and the harder basal part. Is the pith in- 
volved? Are the medullary rays occupied? Where are the 
bacteria located outside of vessels, i.e., within cells or between 
them? In the leaves do the bacteria make special use of the 
vessels? Is the parenchymatic tissue killed in advance of its 
occupation? Parts long diseased are hard to embed on account 
of entrance of air; you will, therefore, collect unshriveled soft 
stems and leaves, and fix without delay, using the air pump. 

Variability. — ^Little is known. 

Transinission. — Nothing is known respecting carriers of the 
bacteria or the natural methods of infection. The organism 
enters readily through wounds and probably also through stom- 
ata and lenticels. Settle the latter by experiments, if you have 
the opportunity. As in fire-blight of the pear, the copious 
ooze of bacteria to the surface of the blighting leaves and 
shoots afTords abundant material for infecting other parts of the 
same tree and for transmission to neighboring trees, and it is 
advisable, therefore, to remove all blighting branches promptly. 

LITERATURE 

For earlier notes by the w^riter read: Science, N. S., Vol. 
XXXI, May, 1910, p. 792; and Phytopathology, Vol.4, 1914, p. 34. 

For Macchiatti's paper consult Malpighia, Anno. V. Fasc. 
VII-XII, Genoa, 1892, pp. 299-303. 

For Boyer and Lambert's paper consult Cojnptes Rendus 
hehd. des Se de. I' Acad, des Set., Paris. Tome CXVII, Aug. 21, 
1893; pp. 342-343. 

Read Doidge's paper "The South African Mulberry Blight" 
{The Annals of Applied Biology, July, 1915, p. 113). 

See also "Etudes sur les maladies du Murier en 1913" by G. 
Arnaud and Ch. Secretain, Annales du Service des Epiphyties. 
Tome II, Memoires et Rapports, Ministere de L' Agriculture, 
Paris. Librairie Lhomme, 1915, pp. 233-249, 11 text figs. 

Consult also "Bacteria in Relation to Plant Diseases," Vol. 
II, 1911, Figs. 3 and 30. 



XII. FIRE-BLIGHT OF APPLE, PEAR, QUINCE, ETC. 

(Called also pear blight, apple blight, quince blight, etc.) 

Type. — ^Fire-blight, so called since the time of Wm. Coxe 
(1817), is a time-hmited, rapid, parenchymatic decay, chiefly of 
the pear, the apple, quince, and other pome fruits (Figs. 276, 277, 
278). From stone fruits it was first described in 1902 by L. R. 
Jones who found it on the cultivated plum (Prunus sps.). It 
was also found independently on the plum by Merton B. Waite. 
It has been seen on the loquat (Eriobotrya), and on the cherry. 
It occurs also on certain wild genera, e.g., Crataegus, Amelanchier, 
Heteromeles (Waite). O'Gara has reported it from the apricot 
in the Northwest. Heald also has reported finding it on the 
apricot in Texas. In 1915 the writer cross-inoculated it to 
apricot readily. He also saw it escape naturally from inoculated 
pears to a neighboring apricot (Fig. 279) and with the organism 
plated from a dying apricot twig produced typical blight on 
pear shoots. Munn has recently reported it as inoculable into 
the blossoms of the strawberry {Phytopathology, vol. 8, p. 33). 
Can you find it occurring naturally on the strawberry? Search 
in the vicinity of blighting pear and apple orchaids. 

It begins by destroying the blossoms, green fruits, and young 
shoots, including young leaves which are specially favorable 
places for its development (Fig. 280), but it passes quickly 
downward, chiefly by way of the bark parenchyma, into the 
inner bark of the larger branches and of the trunk, which often 
are girdled and killed. It gets its common name from the con- 
spicuous black or brown appearance of the blighted branches, 
the dead persistent leaves of which look as if scorched (Fig. 278). 
The blackening of the leaves is a host reaction and occurs in the 
absence of the bacteria, but the bacteria often attack leaves 
as well as stems, passing from the stem through the petiole into 
the leaf blade (Figs. 280, 281). As the fruits ripen and as the 
inner (living) bark of the shoots becomes firm, in late summer or 

3.59 



360 



BACTERIAL DISEASES OF PLANTS 



earl}' autumn, the blight ceases to spread, and the organism, in 
a majority of cases, dies out of the blighted (killed) trunk and 
limbs, but in a variable per cent of cases the bacteria persist in 
certain patches, forming what ]Mr. Waite, who discovered it, 
has termed ''hold-over blight," and Prof. Whetzel "cankers." 
From these spots (Figs. 282 and 283), which ooze living and 




Fig. 276. — Fire-blight on a pear tree. Healthy and blighted branches, seen close. 

:Maryland. July 1, 1914. 

virulent bacteria, especially during the increased sap-flow of the 
spring, and which are visited by bees and other pollen -gathering 
and nectar-sipping insects, the exudate being sweetish, accord- 
ing to O'Gara, the bacteria are carried to the blossoms of neigh- 
boring trees and a new outbreak is started, the organism, brought 
by these insects, growing first in the nectar of the flowers or in 



FIRE-BLIGHT OF APPLE, PEAR, ETC.: TYPE 



361 




YiG. 277.— Branch of an apple tree showing bhghted flowers, fruits and shoots 
due to Bacillus amylovorus. Time of year may be judged from the size of the 
green apple below. Season of 1914. 



362 



BACTERIAL DISEASES OF PLANTS 



•^.y 














Fig. 278. — A pear tree showing limbs recently blighted bj^ Bacillus amylovorns 
(from hold-over blight) and at X the old blight, i.e., that of the previous season. 
Washington, D. C. 1914. 



FIRE-BLIGHT OF APPLE, PEAR, ETC. I TYPE 



363 



bitten or punctured shoots. Sackett believes that we have 
greatly underestimated the number of cases of hold-over blight, 
especially on the smaller limbs and twigs, since in a total of 
83 blighted pear twigs, examined by him in the winter and spring 
of 1910 and 1911 in Colorado, 25 per cent contained living 
blight bacteria. These cultures were taken at the border line 
joining sound and blighted tissues. On the contrary, working 




Fig. 279. — Fire-blight on apricot. A natural infection from an inoculated pear 
tree in one of our hothouses. Photographed .June 7, 1915. 

in Pennsylvania on pruned branches allowed to lie on the earth, 
Fulton found that the bacteria were dead in nine-tenths of them 
at the end of a week (35 branches tested). More tests should 
be made in various localities. Here is a good opportunity for 
useful experiments 

Secondary infections are very common during the growing 
season, owing to the abundant and fluid nature of the bacterial 



3G4 



BACTEEIAL DISEASES OF PLANTS 




Fig. 280. 



FIRE-BLIGHT OF APPLE, PEAR, ETC. : TYPE 365 

slime which oozes through natural openings (stomata and 
lenticels) to the surface of stems, fruits (Fig. 284) and leaves 
(Fig. 285) in great abundance, where it is visited by insects, and 
from which is also drips readily to other parts of the tree 

In the shoots it is primarily a bark disease (Figs. 286, 287). 
It disintegrates the green fruits by multiplying in the inter- 
cellular spaces and dissolving the middle lamellae (Fig. 288) and 
is enormously abundant in such fruits. 

In cultures I have not observed rapid loss of virulence. A 
strain carried along on media in my laboratory for 7 years 
(1908-1915) blighted pear shoots readily, even of a so-called 
''blight-proof" sort, when inoculated by needle pricks in June, 
1915, Another strain on media 5 years was infectious in 1920. 

Fire-blight occurs destructively" every year in some part of 
the United States (Fig. 289), and in certain years (as in 1914 and 
1915) sweeps over the whole country. It has been known for 
a hundred and forty years in the eastern United States where 
it was probably first present on wild shrubs, but its distribution 
in other parts of the world remains uncertain. It has, how- 
ever, been reported from Italy. I have been told also that it 
now occurs in northern Japan on pear and apple, especially on 
the Red Astrachan (Gentaro Yamada). In Cornwall, England, 
I saw what looked at a little distance like fire-blight on apple 
twigs, but on going into the orchard I found that the twigs had 
been smothered by lichens. 

The disease can be controlled by prompt, intelligent and 
severe pruning (Fig. 290). This is the only remedy known. To 
avoid spreading the disease by means of the pruning tools they 
must be disinfected after the removal of each infected limb. 
The cut surface should also be disinfected (see Part I, page 71). 
O'Gara recommends 1:1000 mercuric chloride in water applied 

Fig. 280. — Blight on pear leaves due to Bacillus amylovorus. Introduced 
to show how petioles and midribs often blight in advance of the leaf-blade. In 
these leaves both petioles were blackened on their surface (except at X) and were 
exuding bacterial droplets from numerous stomata, but enough fluid was still 
passing through their vascular bundles to keep the leaf-blades green and turgid 
except an extremely small portion of the base of B, and a much larger but still 
relatively small area along the midrib of A. The invasion came from the bark 
of the shoot, which was blighted. 



366 



BACTERIAL DISEASES OF PLANTS 




Fig. 281. — Growing shoot of Clapp's Favorite pear inoculated 5 days with a 
pure culture of Bacillus amylovorus plated from an apple twig. Bark blighted 
and exuding drops of bacterial slime. Blight running out on petioles (a, a, a) 
and on leaf-blade (b). Terminal leaves still green and turgid but in another 24 
to 48 hours they would have become brown and shriveled. Lowest visible blight 
at X. Internally the bacteria were traced under the microscope 3 inches farther 
down the stem. May, 1915. The blue-black stripe of blighted bark and the pale 
green of the normal part of the shoot photographed exactly alike, both with Ham- 
mer's double-coated orthochromatic plates and with W. and W. panchromatic 
plates. The eye-contrast was finally obtained on the latter plate by using a green 
color screen. 



FIRE-BLIGHT OF APPLE, PEAR, ETC.: TYPE 



367 



by means of a sponge. It is most easily applied on trees that 
have been trained to a vase shape (Fig. 290). Those trees 
trained to pyramidal form with a single central stem (Fig. 278) 
are often killed by the first attack of fire-blight. 







Fig. 282. — Fire-blight canker on apple (Waite's hold-over blight). Spring 
condition — bacteria exuding and likely to be visited by bees and other pollen- 
gathering and nectar-sipping insects. Bark discolored on the right side. After 
Whetzel. 



Cause. — Pear blight or fire-blight is due to Bacillus amylo- 
vorus (Burrill) Trevisan. The original name. Micrococcus 
amylovorus, was given to it by Prof. Burrill, under the assump- 
tion that it destroys starch but such is not the case. It is a 
white, motile, peritrichiate-flagellate (Fig. 291), non-sporiferous, 
non-odorous, non-acid-fast, Gram negative, non-nitrate-reduc- 



368 



BACTERIAL DISEASES OF PLANTS 
















' .■h.'^^J£*<■ 



Fig. 283. — Bark of an apple tree in spring showing Bacillus amylovorus oozing 
to the surface from a patch of "hold-over" blight. Enlarged 6 times from 
a photographic print furnished by Prof. H. H. Whetzel. Planar by James F. 
Brewer. 



FIRE-BLIGHT OF APPLE, PEAR, ETC.: CAUSE 



369 



ing, rather slowly liquefying (gelatin, not Loffler's solidified 
blood serum), more or less viscid (often quite slimy from 
pear and apple fruits), butyrous on agar and potato (D. H. 
Jones) aerobic and facultative anaerobic (with grape-sugar, 
fruit-sugar, cane-sugar, or malt-sugar) , non-gas-forming, sodium 



hHP'^ ' ' ?• ■ ^ . 








'":1 


■ ■ ■ ■ i 



Fig. 284. — Green pear fruit (Duchess) rotted by Bacillus amylovorus. Result 
of a pure-culture inoculation. Ooze of bacteria through stomata can be seen 
in many places above the central area. Below is a larger amount of bacterial 
ooze from cracks in the vicinity of the needle-wounds. 



chlorid-tolerant, sunlight-sensitive, dry-air-sensitive, rod-shaped 
schizomycete, growing on the surface of agar-poured plates in 
the form of circular, small, entire-margined, more or less 
opalescent white colonies, and in the depths as smaller, some- 
times ringed, fringed or hazy-margined colonies (our pathogenic 



370 



BACTERIAL DISEASES OF PLANTS 



X 



Fig. 285.— Blight- 
ing pear petiole. A 
detail from Fig. 280 
at X, showing more 
distinctly the stomatal 
bacterial exudate (Ba- 
cillus a m ylovor u s ) 
from the blackened 
petiole. On the right 
side at A' the tissue is 
still green and turgid. 



cultures of 1905 — not those of 1915; although 
both were from apple). On thin-sown +15 
peptone-beef agar plates, held at 20°C. the 
homogeneous wet-shining surface colonies 
may reach a diameter of 5 or 6 mm. by the 
end of the sixth day (Fig. 292). Their ap- 
pearance when viewed by reflected and ob- 
lique transmitted light is shown, on Fig. 
293. In tubes of nutrient agar there is a 
good growth the whole length of the stab. 
On gelatin plates the colonies are circular 
and inclined to pile up rather than to spread 
widely. In some gelatins a rather prompt 
pit of liquefaction appears around the colony 
(Fig. 294). The color of inoculated milk 
remains unchanged but after some days a 
soft curd settles, and later this is more or 
less completely digested (Fig. 295^4). If a 
lab ferment is produced it must be very 
sensitive to heat (15 minutes at 57°C.). It 
does not redden litmus milk, but precipitates 
the casein usually within, a few days (4 or 
5). There is often a partial reduction of the 
litmus, the medium being bluer than the 
check at the top, paler blue at the bottom 
(in the curd) and wholly reduced in the 
middle, i.e., changed to a pale brownish 
white. It grows rapidly in bouillon especi- 
ally if cane-sugar is added. In beef bouillon 
or potato broth there is not only a prompt 
and heavy clouding with presence of pseu- 
dozoogloeae (turbidity) but if undisturbed 
a slight granular pellicle forms. Clouds 
potato juice in closed end of fermentation 
tubes without gas. Indol reaction scanty 
or negative. Tolerant of small quantities 
of malic acid and citric acid but not of lactic 
acid. I now doubt if malic acid actually 



FIRE-BLIGHT OF APPLE, PEAR, ETC.: CAUSE 371 

stimulates growth, as Waite supposed. Test. Unneutralized 
acids of gelatin inhibit growth. Tests may be made. Growth 
is best when the gelatin is made neutral or nearly neutral 
to phenolphthalein by use of sodium hydrate. Acids are 
formed from various sugars. In Uschinsky's solution no 
growth, or slow growth, unless peptone is added: grow,th copi- 
ous, not viscid (D. H. Jones). In Cohn's solution no growth 
or slight (Repeated in 1915 with the same result). The opti- 
mum temperature is 30°C. Growth at 3°C. is very slow 
(D. H. Jones), and there is no growth at 0.5°C. Exposure in 




Fig. 286. — Cross-section of a young pear sliuot showing cavities in the bark 
due to Bacillus amylovorus. The bacteria diffuse out of such cavities very readily 
on staining. Inoculated by the writer in May, 1915. 

beef broth at 43°C. for 10 minutes retards growth and at 43.7°C. 
kills (L. R. Jones). The thermal death point in bouillon lies 
between 45° and 50°C. (D. H. Jones). In liquids all are killed 
by ten minutes' exposure to 55°C. (O'Gara). In a recent 
inoculation (Oct., 1919) using our 1915 isolation from apple, the 
organism clouded at 43°C. (but with retardation) and finally 
at 44°C. One also of the four tubes exposed at 45°C. clouded 
(after 6 days) but none at 48°C. (15 days), when exposed 
for 10 minutes in the water bath in -|-15 peptone beef bouillon. 
The checks grew promptly and the six uninoculated tubes re- 



372 BACTERIAL DISEASES OF PLANTS 

mained sterile. In a repetition of the experiment there was no 
clouding either at 45° or 46°C. As a rule it lives long on culture 
media. Drying on cover glasses up to 5 days has no appreciable 
effect (L. R. Jones, D. H. Jones). Tolerates HCl in bouillon 
(+4) up to +16 and NaOH down to -6 (D. H. Jones). Opti- 
mum reaction for growth + (D. H. Jones). It resists freezing 
fairly well. In tests made in 1919, 15 per cent survived. It 
is identified readily by its behavior under bell jars, when streaked, 




Fig. 287. — Pear blight: A detail from one of the cavities shown in Fig. 286. 

on raw green, pears, which should rot; and on ripe ones, which 
should not rot (Waite's method). This test may be made also 
in late winter or early spring (in advance of the growing season) 
on vigorous shoots of the pear by bringing them into a warm 
room and standing the lower parts in a jar of water until they 
have begun to sprout (Waite's method, verified by Katherine 
Golden). Then with a sharp knife remove the tops making a 
slant stroke, on which should be rubbed the organism to be 
tested. If it is a virulent strain of Bacillus amylovorus, the 



FIRE-BLIGHT OF APPLE, PEAR, ETC. I CAUSE 373 

shoots will soon begin to show the blight. To keep the cut sur- 
face from drying out, the jar of water should be covered with a 
tall bell jar; it should also be protected from direct sunshine. 
The shoots should be of sensitive varieties, e.g., Clapp's Favorite 
or Flemish Beauty, and the organism must be virulent. 

The following unsupported or wholly erroneous statements 
respecting the pear blight organism have gained more or less 




ly.^ 



Fig. 288. — Section from a disintegrating pear fruit (like Fig. 284), showing 
Bacillus amylovorus between and in the cells. Photomicrograph by the writer. 
X 1000. 

currency, viz., that it is a Micrococcus, that it is non-flagellate 
(the flagella are hard to stain), that it is a rapid destroyer of 
starch, that it produces gas and more specifically carbon dioxide 
or hydrogen, that it produces butyric acid, that it will not grow 
on agar, that it does not liquefy gelatin, that it is yellowish, 
that it is red or reddish, that it cannot be found swarming 
in the leaves, that it causes a disease of peach trees and of Lom- 
bardy poplar trees, that it commonly passes the winter in dead 
limbs or in the earth, that it cannot be cross-inoculated from 
apple to pear and quince or vice versa, that it never enters the 



374 BACTERIAL DISEASES OF PLANTS 

plant in the absence of wounds. Many other misconceptions 
exist among horticulturists and the uninformed multitude, e.g., 
that the disease is due to "thunder and lightning" or to ''frozen 
sap," but the above include about all that the writer has observed 
in the writings of scientific men. 

Technic.^ — Knowing the biological peculiarities of the pear- 
blight organism, isolation is not difficult if one attempts it only 
from freshly blighted fruits or shoots and makes his transfers 
only from the advancing margin of the diseased parts, using the 
methods described under No. I. Then usually the poured 
plates are pure cultures. Isolation from older blight is more 
difficult, and often it is impossible owing to the prompt death 
of the organism in the blighted dry tissues. The commonest 
invaders on the plates are non-parasitic yellow colonies and 
sometimes only these appear. 

For inoculation purposes, immature pear and apple fruits are 
very convenient since they are everywhere available in May and 
June. They may be inoculated by needle stabs or other wounds 
at any stage of growth preceding that internal change which 
takes place when they have reached full size and begin to ap- 
proach maturity — fruits one-fourth or one-half grown are very 
suitable. They may be inoculated either on the tree, which is 
the more natural way, or under bell jars, especially if they are 
sliced. In the former case they must be protected from insect 
visitation by covering with a double fold of mosquito netting 
or with surgeon's gauze. 

For inoculations on shoots, it is convenient to have half a 
hundred small pear trees in pots, some of which must be growing 
freely. Others which are growing feebly should, however, be 
inoculated for contrast. 

Slow-growing and rapid-growing shoots on various kinds of 
pear trees in the open may also be inoculated (the best time is 
May- June) but they should be covered with netting to keep off 
insects and thus avoid the spread of the disease. 

In the same way if the blossoms of pear, apple or quince are 
inoculated, which may be either by means of an atomizer, a 
platinum needle, or a pipette drawn to a fine point, the clusters 
must be covered, unless it is desired to study the transfer of the 



FIRE-BLIGHT OF APPLE, PEAR, ETC. : TECHNIC 



375 




376 



BACTERIAL DISEASES OF PLANTS 




Fig. 290.— Photographs from a Maryland pear orchard showing result of 
proper eradication of pear Wight. Views made in March, 1912. Blighted limbs 
removed 6 years earUer. From photographs by Merton B. Waite. 



FIRE-BLIGHT OF APPLE, PEAR, ETC.: TECHNIC 377 

disease by insects. Avoid wounding the flowers in making the 
inoculations. 

For varietal contrast, Seckel, Duchess, Douglas, Winter 
Nelis or Kieffer (which blight slowly) may be compared with 
Bartlett, Howell, Flemish Beauty or Clapp's Favorite (which 
blight rapidly). There is also a so-called "blight-proof" pear 
derived from the Chinese Sand pear, which is not^blight-proof 




Fig. 291.- — Flagellate rods of Bacillus amijlovorus. Stained by van Ermen- 
gem's silver nitrate method. Isolation of 1908 from apple. X 1000. 

An isolation of 1915 from apple stained by Miss Bryan also showed peri- 
trichiate flagella of the same type. 

(Fig. 296), and which may be tested. No entirely resistant 
sorts are known to the writer but recently Reimer has discovered 
several. These are stocks of Pyrus ussuriensis and other Asian 
sorts (collected in China by Frank N. Meyer and by F. C. 
Reimer). These are now being tested on a large scale in the 
open field in several localities in the West by Reimer, and in the 
East by the United States Department of Agriculture. O'Gara 



378 



BACTERIAL DISEASES OF PLANTS 



states that stocks of Kieffer and Winter Nelis on the Pacific 
Coast are quite resistant, the bhght on sensitive varieties often 
stopping at the point of union when these stocks have been used. 
Out of season the only means of obtaining material suitable 
for inoculation is by the use of a forcing house, by use of young 




Fig. 292. — Surface and buried colonies of Bacillus amylovorus from agar 
poured plates, (a) The buried colonies have vague, fuzzy margins. X 7. Age, 
4 days. Plated from an apple, in 1905. It was with descendants of this isolation 
that Dr. Rudolph Aderhold, of Berlin, obtained his infections. From the unlike 
appearance of the buried and surface colonies he thought at first, so he told me, 
that I had sent him a contaminated culture. (6) Plated from an apple limb in 
1915. Buried colonies not fuzzy but also infectious (see Figs. 281, 296). 

seedhngs, or on cut shoots by the simple means described under 
Type, which, however, sometimes fails. 

As material for inoculation, streaks on agar or potato may be 
used, or bouillon or potato-broth cultures. 



FIRE-BLTGHT OF APPLE^ PEAR, ETC. I TECHNIC 379 










• 




Fig. 293. — A. Surface and buried colonies of Bacillus ainyloporus (isolated 
from apple) on a +14 beef-peptone agar plate. Photographed February 5. 
1919, by direct transmitted light. X 10. 

B. From the same plate but photographed by oblique transmitted light. X 10 



380 BACTERIAL DISEASES OF PLANTS 

Determine 

For the organism. Morphology. — Size in microns (when 
growing rapidly in media the rods are often several microns 
long — 3 to 6 or more), form, aggregation of elements, i.e., chains, 
filaments, pseudozoogloeae, etc., motility (on margin of hanging 
drop), absence of endospores, presence and distribution of flagella 
(use V. A. Moore's modification of Lofiier's flagella stain, or 
van Ermengem's stain). Try Gram's stain; acid-fast stain; 
Capsule stains. Do involution forms occur? 

Cultural Characters. — Appearance of colonies on thin-sown 
agar plates (Figs. 292, 293) and on gelatin plates; in stabs and 
streaks on agar, ditto on gelatin; behavior in peptone bouillon, 
in potato broth; try also malated and sugared broths. Growth 
in nitrate bouillon, Cohn's solution, Uschinsky's solution, milk, 
litmus milk. Is a lab ferment produced in milk? Behavior in 
peptone water in fermentation tubes with various sugars, alco- 
hols, and acids. Try it also in fermentation tubes with potato 
broth and other plant juices, e.g., apple or pear broth. Is there 
any clouding in the closed end? Test milk in fermentation 
tubes. Can you get the results shown in Fig. 295? Determine 
the nitrogen nutrition of this organism. 

Non-nutritional Environment. — Maximum temperature for 
growth; minimum? Thermal death-point? Effect of sunhght; 
of dry air (killed quickly), of freezing (salt and pounded ice), 
of salted bouillons, of chloroform in bouillon, of acids, of alkali, 
of germicides. Read Reimer's papers and experiment with vari- 
ous germicides. 

For the disease. Signs. — Period of incubation (examine 
morning and night each day); signs in flowers (especially in 
early stages of the disease — first 72 hours) ; in shoots (observe 
that the bark of the shoot may be entirely blackened on the sur- 
face from the bacterial action and yet for a time the terminal 
leaves may remain green and turgid. Why?); on leaves: a, di- 
rect effect, i.e., dark lines running out along the petiole, midrib 
or side veins, due to bacterial infection from the shoot; b, in- 
direct effect — black specking, flagging, reddening or browning, 
due to stem injuries farther down. Note the persistence of the 



FIRE-BLIGHT OF APPLE, PEAR, ETC.: SIGNS 381 

leaves. Learn the appearance of ''hold-over blight." Usually 
on smooth trunks such patches may be detected readily by the 
practised eye in the absence of the exudate since their color is 
somewhat different from that of the normal bark — redder, 
browner. Under rough bark detection is more difficult and a 
gouge should be used. Is the dark color of the blighted leaves 
and shoots a bacterial stain or a host reaction? Kill leafy pear 
shoots in various ways and see what results follow in the leaves. 
To what extent are the roots attacked? Write a description of 
the disease. 




Fig. 294. — Buried and surface colonies of Bacillus amylovorus after 3 days at 
21°C. on +10 peptone beef gelatin. The bulk of the surface colony is floating 
in the middle of a pit of liquefaction. Isolated from apple in 1915. Photographed 
by the writer. X 7. 

Histology. — How many centimeters in advance of visible 
blight can the bacteria be traced down the blighting shoot? 
What does this teach you relative to pruning for removal of the 
disease? Is the organism motile in the tissues? Is the wood 
attacked or only the bark? To determine this, examine in 
various places from the soft extremity of the blighted shoot 
downward. Any differences (compare with No. XI)? Gara, 
who has had much experience, states that the bacteria may 
occur in the rich sap wood of the Bartlett, Howell, and other 
pears and in that of the Spitzenberg apple. According to 
Reimer this apple is no longer planted in South Oregon because 
of its great susceptibility to blight. When the bark only is 



382 



BACTERIAL DISEASES OF PLANTS 



involved which suffers most, phloem or cortical parenchyma? 
Is the cambium also attacked? What is the color of the nner 
bark in patches of "hold-over" bhght? What tissues are 
honeycombed with bacterial cavities? When the oro;anism 




Fig. 295. — A. Fermentation tube of milk containing Bacillus umijlovorus. 
Col. 6, isolation of 1915 from apple. Inoculated August 18, 1919. Photographed 
Sept. 27. Milk cleared and curd digested in closed end. B. Check tube. 

runs out into leaves what petiolar tissues does it invade? 
In petioles I have observed it wedging apart cells of the bark 
parenchyma and also forming cavities in the xylem part of the 
vascular tissues. 



FIRE-BLIGHT OF APPLE. PEAR. ETC.: HISTOLOGY 383 

If possible, embed and section on the 
microtome early stages of blossom blight. 
Can you make out: a, multiplication of 
bacteria in the nectary: b. invasion of the 
ovary and pedicel? Is there any choice 
in the tissues invaded? Study also bhght- 
ing shoots and fruits. The bacterial slime 
is often abundant enough to drip from the 
hands when such fruits are handled after 
cutting. Is the organism always inter- 
cellular or does it sometimes also enter the 
cells? There is a difference of opinion on 
this point. Do not decide too hastily. Is 
the cell-wall destroyed? ^Miat becomes 
of it? Does the organism commonly come 
to the surface on attacked stems? on 
fruits? ]\Iake permanent stained prepara- 
tions showing relation of the organism to 
the various tissues. Contrast with No. I. 

Variability. — How long does an at- 
tacked shoot live? Using a susceptible 
variety, study effect on rapidity of blight 
of: a. rapid vs. slow growth, which may 
be correlated with time of year (]\Iay- 
June vs. August-September) ; b, amount of fig. 29G.— Shoot of 
rainfall or water given: c. moderate vs. high -Blight-proof pear ihy- 

temperatures: d. light vs. heaVV inocula- brid between the Chinese 
^ ' . .Sand pear and some coiu- 

tions, €, kind of cultivation and manuring. ^^^^ pear). Inoculated 
Rich soils and alkali soils are said to favor 5 days with a pure cul- 
the development of the disease (O'Gara). ^u^e of Bacillus am yhv- 

_, , . , orus plated m 191.5 from 

If you have opportunity to study ^^ ^^i^^^^^^^ ^ppl^ ^,,^^,h 
blighting orchards look for varietal differ- Bark blighted (.browned) 
ences. Some commonly cultivated pears and beads of bacterial 

, . ' .1 T^ 1 slime oozing from thein- 

are nearlv immune, e.g., the Douglas, a ^ . .^ k + „ «+„ 

^ o ' tenor through stomata. 

seedhng of the Kieffer (Y. B. Stewart); May. 1915. This variety 
others are verv susceptible. The same is bUghted rather freely as 
true of apples.^ Make inoculations on re- ^^^ '•^"it «f "^^^le prick 

f^ ... moculations. 

sistant and susceptible varieties and record 
the results. 



384 BACTERIAL DISEASES OF PLANTS 

Whatever you do, make full and legible notes. 

Transmission. — On account of its large dependence on ani- 
mals (chiefly on insects) for distribution, this is one of our most 
interesting diseases. It is quite easy for any student who has 
the organism and a blossoming apple or pear tree to start blos- 
som-infection and demonstrate transmission of the disease by 
bees. This was discovered by Waite. According to O'Gara 
the disease may be transmitted by at least 50 kinds of insects 
visiting pear flowers. 

The disease is common, however, on nursery stock not in 
blossom, and here bees and flies are probably not the common 
agents of transmission. Sometimes birds carry the germs on 
their bills (sap-suckers) or on their claws, which often break and 
scratch twigs. O'Gara believes that the disease may be in- 
troduced sometimes through growth cracks (see No. XIII). 
D. H. Jones has shown that aphides by their punctures may 
transmit the bacteria, especially on apple trees. Some beetles 
also are carriers: Scolytus (D. H. Jones); and some bugs other 
than aphides: Lygus (V. B. Stewart). More recently (1915), A. 
C. Burrill also has proved the disease to be transmitted by 
aphides (A.avenae), and by an apple leaf-hopper (Empoascamali). 
The disease may also be spread by means of pruning tools. 
Waite saw a nursery block of 10,000 Bartlett trees destroyed by 
pear blight which was transmitted in the Spring on pruning 
tools. There was some '^ hold-over" blight in the nursery and 
when the tops of the trees were cut down to the dormant inocu- 
lated buds the tools were contaminated and the blight was 
distributed to nearly every tree. In the West, on the apple, it 
has been found to enter through wounds made by crown galls 
(O'Gara). It may also enter and blight trunks through soft 
water-sprouts which for this reason should not be allowed to 
grow around the base of the tree. According to O'Gara 80 per 
cent of the initial fire-blight infections in California and South 
Oregon were through water-sprouts and low fruit-spurs. Heald, 
in Washington State, has found the disease entering the plant 
(apple and pear) commonly through the leaves as if by water- 
pore or stomatal infection (1915). Do apple and pear leaves 
have water pores? Section a leaf-tooth and see. V. B. Stew- 



FIRE-BLIGHT OF APPLE, PEAR, ETC.: TRANSMISSION 385 

art (1915) showed that it may enter through wounds made by 
hailstones (compare with No. XIII and XIV). 

The disease is probably carried sometimes on nursery stock, 
but not often, I think, owing to the fact that it dies out rather 
quickly in the dead shoots. Probably this fact also accounts for 
its not having been introduced into other parts of the world, if we 
may assume, as seems probable, that the eastern United States 
is the original home of the disease, and that it has not occurred 
until recently in any other pear or apple-growing region of the 
world. Was it introduced into Japan from the United States? 

Eradication of the Disease 

Complete freedom from fire-bUght may never be hoped for, 
any more than from any other widespread and highly infectious 
disease, but there are certain palliatives which if properly applied 
will reduce its destructiveness to insignificant proportions. 
These fall into two main categories: (1) tree surgery; (2) resis- 
tant varieties, or rather immune stocks for sensitive varieties, 
since the latter include all our finer sorts of pears , and apples, 
the discarding of which is not to be thought of. 

It has been demonstrated conclusively by Waite and others 
that the spring blight is distributed principally by bees and other 
insects which obtain the infection from oozing patches of "hold- 
over" blight. In the control of this disease it is, therefore, of 
prime importance to search the trunks, limbs and roots in late 
autumn or winter and remove all blighted spots. Such removal 
is equally important if the blight is wind-distributed, as main- 
tained by Stevens and his associates {Science N.S., Vol. XLVIII, 
pp. 449-450). If this is done thoroughly over a wide area there 
will be very little spring-blight. 

If the eradication of the canker, or hold-over blight, has been 
neglected the next best thing is to cut out the spring- and summer- 
blight thoroughly as fast as it appears, including the neglected 
cankers, disinfecting the tools from limb to limb and tree to 
tree, since if you neglect this you will inevitably distribute the 
bacteria by means of your saw, gouge, knife and p uning shears. 
Both tools and tree-wounds should be disinfected. Mercuric 

25 



386 BACTERIAL DISEASES OF PLANTS 

chlorid, otherwise known as corrosive sublimate or bichlorid 
of mercury, is generally recommended for this purpose. It is 
made up in the proportion of one ounce of the poison to one 
thousand ounces of distilled water, clean rain, water or boiled 
well water. This may be applied as O'Gara recommends by 
a sponge attached to the wrist or to the buttonhole bj^ means of 
a string about 2 feet long. The effectiveness of this germicide 
is destroyed bj^ contact with a metal container. It must be 
kept, therefore, in glass bottles or wooden pails, never in tin 
pails. Tools ma}^ also be dipped into 5 per cent carbolic acid 
water or into 1 part of formalin diluted with 9 parts of water. 
The hands must be kept out of both substances. For notes on 
Reimer's newer germicides for fire-blight, consult Part I, page 71. 
Eventually in this country the pear blight problem, which 
is a very serious one, will be solved by discarding altogether the 
susceptible French seedling stocks (Pyrus communis) on. which 
most of our valuable sorts are now worked, and substituting 
resistant stocks. The most hopeful substitutes are certain East 
Asian species, notably Pyrus ussuriensis, P. ovoidea, P. calleryana, 
and P. variolosa. All of these species are very resistant to 
Bacillus amylovorus, and the main question now appears to be 
which are the most resistant, and which will prove most satis- 
factory in other respects, i.e., grow equally with the graft, giv- 
ing to it a firm union and a long life. For details the reader is 
referred to F. C. Reimer's very interesting paper "Blight 
Resistance in Pear Trees and Pear Stocks" (1916 Ann. Rept. 
Pacific Coast Assoc, of Nurserymen. Also a separate pp. 1-8). 
More recently (Jul}^ 3, 1919) Mr. Reimer, who is superintendent 
of the Southern Oregon Branch Station of the Oregon Agricul- 
tural College Experiment Station (Post Office Talent, Oregon), 
has written me as follows: 

"In my pamphlet I made the following statement regarding Pyrus ussurien.ns: 
'This species appears to be immune to pear blight, at least under the conditions in 
Southern Oregon.' 

"This statement was based on inoculation work performed with only a very 
limited number of wild forms of this species. Since that statement was published 
we have collected at this Station a very large number of types of this species and 
have repeatedly inoculated them with the pear blight organism. The results based 
on four seasons' work may be summed up as follows so far as Southern Oregon is 
concerned : 



FIRE-BLIGHT OF APPLE, PEAR, ETC.: ERADICATION 387 

"1. Pyrus ussuriensis is more resistant to pear blight than any other known 
species of Pyrus. 2. Many forms of Pyrus ussuriensis have so far proved immune 
to pear blight. faiUng to bhght. even in the tips of the j^oung, tender, vigorous 
shoots, when inoculated. 3. Some forms of Pyrus ussuriensis blight only in the 
young tender shoots when inoculated, the blight usually killing such shoots back 
only from one to ten inches, and very rarely as much as fifteen inches, in case of 
extremely vigoi'ous trees. In such cases the disease is usually confined to the cur- 
rent season's growth, although very rarely and in the most extreme cases it slightly 
enters the previous season's wood. The disease in these forms has always been 
confined to that portion of the branch less than one-half inch in diameter, and very 
seldom has progressed into wood more than one-fourth inch in diameter. 

''These results were obtained in a region where pear blight is extremely viru- 
lent, on very fertile, irrigated and thoroughly cultivated soil, which produces a 
very vigorous growth, and where every effort has been made to induce the trees 
to blight. Up to the present time no natural infections have ever been found on 
the young trees of Pyrus iissuricnsis." 

LITERATURE 

For literature, consult various writings of Burrill, Arthur, 
Waite, Whetzel, L. R. Jones, D. H. Jones (Bull. 176, Ont. Ag. 
Col.), Stewart (V. B.), Sackett, Bachmann, Fulton, Heald, 
O'Gara, Merrill, Reimer, Stevens, etc. 

See also "Bacteria in Relation to Plant Diseases," Vol. I 
(1905), plates 28-31, and Fig. 61. For various brief notes on 
the organism see also the index to Ibid., Vols. I, II, III. 

The student should learn early how to use literature and 
should be a wide and eager reader not only of all the newer 
things but also of old books and papers. Search out all the 
pear-blight papers from the above hints and make a respectable, 
chronological bibliography. 

Aderhold's note is in Aderhold and Ruhland's paper on 
"Die Bakterienbrand der Kirschbaume," Arb. a. d. Kaiserl, 
Bio. Anstalt f. Land. u. Forstw., V Bd., 6 Heft, pp. 334-336. 

Reimer's paper "A new disinfectant for pear blight" is 
in Monthly Bulletin of the [Cahfornia] State Commission of'Horti- 
culture, Vol. VII, No. 10, Sacramento, Oct., 1918, pp. 562-565. 

P. J. O'Gara's paper "Pear Blight and Its Control upon the 
Pacific Coast" was published in the Medford ]Mail Tribune, 
Medford, Oregon, 1910, and a separate was issued, 8 vo., pp. 1-34. 

A pear grower's observations on the disease as it occurred 
in New Jersey in the first years of the 19th century may be found 
in Wm. Coxe's book. Who was Coxe? 



388 



BACTERIAL DISEASES OF PLANTS 



The name Micrococcus amylovorus was published by Prof. 
Burrill in 1882. M. amylivorus is a typographical error of 1883. 




Fig. 296''. — Illustrations of Pyrus ussuriensis Maxim. Resistant to fire-blight. 

1. Resetted terminal growth at close of season. 2. Leaf showing bristled 
serratures (% nat. size). 3. Fruit, showing reflexed persistent calyx lobes iJ4 
nat. size). 4. Ripe fruit cut open, showing easy separation of core. 5. A cul- 
tivated varietur, called Man Yuan Hsiang, (about % nat. size). All from 
photographs by F. C. Reimer. 



XIIL THE OLIVE TUBERCLE 

(Syn. Olive knot) 

Type. — This disease is a conspicuous overgrowth. It occurs 
on wild and cultivated olives, forming large or small, irregular, 
spongy or cheesy knots or excrescences (Figs. 297, 298) which 
decay rather quickly. Attacked limbs are dwarfed or killed, 
and occasionally' the whole plant is destroyed, particularly if small 
and on irrigated land, but more often the trees are only stunted 
and rendered unfruitful. New outgrowths often occur around 
the old dead knots, and also similar growths at a distance from 
them. Roots, trunk, branches, and leaves, are subject. Often 
when a terminal shoot is attacked it ceases to grow, even if 
it had been very vigorous (Figs. 299, 300), and the branch 
is continued by one or more of the lower side shoots, the terminal 
shoot dying. Once attacked a tree seldom or never recovers, 
that is, the disease persists from year to year, and invades new 
shoots and lower parts of the old (Fig. 301). 

No tumor-strand occurs and the secondary tumors have the 
structure of the tissue in which they are lodged, i.e., the disease 
is a granuloma. The bacteria are abundant and easily visible, 
being lodged first between the cells and eventually in intercellu- 
lar, irregularly branched cavities, around which the tissue often 
has a water-soaked or brownish appearance (Figs. 302, 303). 
During rainy weather the bacteria ooze readily to the surface 
of the tumor in great numbers (Home) whence they are washed 
to other parts of the same tree and carried probably to other 
trees, entering through wounds to form other tumors. Deep 
tumors may also arise at a distance from the first tumor and 
these are due to bacteria which have migrated from the primary 
tumor by way of the spiral vessels of the inner wood which in 
such cases are browned, more or less disorganized, and occupied 
by the gray- white slime of the bacteria (Figs. 304 X, 305). The 
knot contains or may contain both wood and bark, the vessels 

389 



390 



BACTERIAL DISEASES OF PLANTS 




Fig. 297. — Olive branches from Genoa, Italy, bearing tumors due to Bacterium 
samstanoi. Collected for the writer by P. J. O'Gara in 1905. 



THE OLIVE tubercle: TYPE 



391 



being greatly distorted and reduced in number while the paren- 
chyma is in excess as in crown gall. Often a large part of the 
tumor, as in Fig. 306, is composed of bark, and then the tumor 
is of a soft ''cheesy" character. The structure of a young tumor 
developing on the under surface of a leaf is shown in Fig. 307. 





Fig. 298. Fig. 299. 

Fig. 298. — Pure-culture inoculations of Bacterium savastanoi in olive shoots 
in 1903. Left-hand shoot Gentile, others Nevadillo Blanco. Time, 57 days. 
The organism used was plated from a California olive knot. J-^ nat. size. 

Fig. 299. — Pure-culture inoculation of Bacterium savastanoi made in 1910, 
on an olive shoot at X, which became dwarfed and died. The lower tumors are 
later surface infections derived from bacteria that oozed from X. Photographed 
in 1912. ~i nat. size. 



This disease has been known since the time of Theophrastus 
(Savastano) and occurs in all olive-growing regions around the 
Mediterranean, and also in California, Argentina (Haumann- 
Merck) and other parts of the world. 

This same disease, or a very similar one, occurs on the 
European ash {Fraxinus excelsior) in France, Germany, Austria 



392 



BACTERIAL DISEASES OF PLANTS 







Fig. 300.— Olive tree from one of the Department of Agriculture pathological 
hothouses showmg result of an inoculation of Bacterium savasta?ioi (at X). Ter- 
mmal shoot stunted and many secondary infections, due to slight (natural) wounds 
and the free use of the gardener's hose. The artificial inoculation was made 
November 1, 1910, and most of the lower tumors developed the followin«^ year 
Photographed January 31, 1912. 



THE OLIVE tubercle: TYPE 



393 




and Italy. Noack in Germany described this ash disease in 
1893 and attributed it to bacteria, solely on the basis of his micro- 
scopic examinations. Subsequently Vuillemin claimed it to be 

the same as the olive tubercle with- 
out, however, giving his reasons. 
The writer first saw the ash disease 
in the vicinity of Vienna in 1913. It 
persists from year to year on the 
trunk and limbs, attacking chiefly 
the bark, making rough cankerous 
thickened patches as large as one's 
hand or larger, but also stimulating 
the growth of the wood so that the 
cankered part of the trunk or branch 
may be twice the diameter of the 



^^ 









Fig. sol Fig. 302. 

Fie. SOL — Olive tubercle. A detail from Fig. 300, showing secondary surface 
infections. The primary inoculation was in 1910 with a pure culture isolated 
from material collected by Florence Hedges at Portofino, Italy, in 1910. Photo- 
graphed December 5, 1912, department of Agriculture hot-house. Tubercles 
6 to 12 months old. One-half natural size. 

Fig. 302. — Cross-section of a young, cheesy olive tubercle (pure-culture in- 
oculation), showing small brown bacterial areas with water-soaked borders. 



normal parts above and below it. The micro-organism present in 
these ash cankers has been studied critically in my laboratory 
by Nellie A, Brown and myself and is scarcely distinguishable 
from the olive-tubercle organism morphologically and culturally 



394 BACTERIAL DISEASES OF PLANTS 

in a variety of media and yet is not absolutely identical. With 
it we have produced typical small cankers on American and Euro- 
pean species of ash, especially on the European Fraxinus ex- 
celsior, but no tumors on the olive although repeated inoculations 
were made on young olive shoots both in 1914 and 1915. The 
ash organism, therefore, should be regarded probably as a 
variety of Bacterium savastanoi rather than as identical with the 
olive organism, or as a distinct species, but further studies and 
comparisons should be made. 

The oleander in Europe and in some parts of the United 
States is also subject to a bacterial overgrowth on leaves and 
shoots, which by various observers has been thought to be due 
to the same organism as the olive tubercle, but my observations 
and inoculation experiments lead me to think it is not due to the 
olive- tubercle organism. This is also Petri's opinion. 

Cause. — In 1886 Archangeli described the olive tubercle, 
giving the name Bacteriuin oleae to something observed in it 
but without what we should now consider to be a proper de- 
scription, i.e., it was named from the microscope, without cultures 
or proofs of its infectiousness by inoculation, and with the state- 
ment that it probably had nothing to do with the disease, which 
was ascribed by him to other causes. Savastano's inoculation 
experiments (1887-1889), repeated and confirmed by Cavara, 
first proved the olive tubercle to be due to bacteria, but neither 
one of these men described the organism sufficiently. Savastano 
called his cultures Bacillus oleae-tuberculosis, but, following Tre- 
visan, systematic writers generally have spoken of Bacillus 
oleae (Arch.) Trevisan as the cause of the olive tubercle. Subse- 
quently Schiff-Georgini isolated and described very carefully 
a non-infectious, white, spore-bearing, peritrichiate, filamentous, 
potato bacillus from the olive-tubercle, called it Bacillus oleae 
(Arch.) Trev., and claimed to have obtained tubercles repeatedly 
by inoculating it into olive shoots (1905). On the contrary, 
Berlese (1905) considered a yellow organism isolated by him 
to be Bacillus oleae (Arch.) Trev., and the cause of one type of 
olive tubercle. Several saprophytes occur commonly in olive 
tubercles, one of which at least is yellow, i.e., that seen by Ber- 
lese. Savastano must also have had a yellow organism in some 



THE OLWE tubercle: CAUSE 395 

of his cultures, since he describes the olive-tubercle organism as 
yellow^on media, which it is not. According to Petri this yel- 
low, capsulate, peritrichiate, liquefying bacillus (which he calls 
Ascobacterium luteum Babes) splits olive oil, forming freely a 
fatt}^ acid, and is more tolerant of acids and of meat extract than 
the olive-tubercle organism. 

There being nowhere in literature any proper description 
of the olive-tubercle organism and in certain quarters much 
scepticism as to the bacterial origin of the growth {vide Robert 
Hartig's Lehrhuch, 1900, p. 211, and Alfred Fischer's Vorles- 




V 



Fig. 303. — Same series as Fig. 302, Init further enlarged to show irregular bacterial 
fissures with water-soaked borders. 

ungen, 2 Aufl., 1903, p. 277), the writer undertook (in 1903) to 
examine the whole question experimentally. For this purpose 
olive tubercles were obtained from Genoa, Italy, and from Cali- 
fornia, and cultures and inoculations in parallel series were in- 
stituted, he results being: (1) repeated demonstration of the 
pathogenicity of a particular organism; (2) the non-pathogenicity 
of all the others, including the common yellow species of bacillus 
and SchifT-Georgini's white species which was obtained from 
Krai of Prague who had received it from Kornauth of Vienna 
to whom it was given by Schiff-Georgini ; and (3) a description 



39G 



BACTERIAL DISEASES OF PLANTS 



of the parasite, which, on account of the reigning confusion 
respecting the nature of Bacillus oleae (Arch.) Trevisan, was 
given a new name Bacterium savastanoi, in honor of Luigi 
Savastano (Fig. 308), who first proved the oHve tubercle to be a 
bacterial disease. With these introductory remarks we may 
proceed to a description of the organism. 







Fig. 304. — Cross-section of an olive petiole showing the brown channel of 
bacterial infection at X. Cut between a primary stem-tubercle (due to needle- 
pricks introducing Bacterium savastanoi) and a deep (unruptured) secondary- 
tubercle on the leaf. See next figure. 

The olive tubercle is due to Bacterium savastanoi EFS. 
This is a slow-growing, white, non-sporiferous, motile, 1 to 4 
polar-flagellate (Fig. 309), aerobic, non-liquef^'ing (gelatin 
and Lo filer's solidified blood serum), non-gas-forming (see 
Petri's statement), non-nitrate-reducing, sunlight-sensitive, heat- 
sensitive, acid-forming (with grape sugar and galactose), 



THE OLWE tubercle: CAUSE 



397 



non-milk-curdling, chloroform-tolerant, sodium chlorid-sensi- 
tive, acid-sensitive (but not to the acid of Cohn's solution), 
alkali-sensitive, slightlj^ viscid (7th daj^ on agar, 3rd day on 
steamed potato), rod-shaped, or occasionally short filamentous 
or catenulate (up to 10m or 15/i, rarel}^ 40//) schizomycete, forming 
on the surface of +15 peptone-beef agar poured-plates slow- 




FiG. 305. — A detail from Fig. 304 in the channel of infection, showing the bacteria. 
Tissues sHghtly out of focus. XlOOO. 

growing colonies, translucent at first then pure white, which 
on thin-sown plates at 22°-23°C. may be 1.5 to 3 mm. in diam- 
eter at the end of the third day (2 to 5 mm. at the end of the 
7th dajO and are circular, fiat, smooth, glistening, and entire, 
or nearly so (Fig. 310), the internal structure under the micro- 
scope being amorphous or fine granular; the buried colonies 
are quite small and often biconvex. Young and perfectly 
smooth surface colonies may also show for a short time a 



398 BACTEEIAL DISEASES OF PLANTS 

reticulate or fish-scale inner structure, or an opaque white center 
with a translucent margin. On gelatin the marginal growth 
of surface colonies or streaks is unlike that of the body of the 
colony, being undulate-erose, frilled, lobed or incised (Figs. 311 
and 312). With 1 per cent dextrose added to the gelatin the 
colonies are frequently ring-marked (Fig. 313) . In + 15 bouillon 
there is a thin clouding but no rim, pellicle or flocculence during 
the first 4 days; later there is or may be a thin pellicle. In 
neutral bouillon no rim or pellicle was observed. In undis- 
turbed tubes of 2 per cent Witte's peptone water after 5 or 6 
days there is a white pellicle which falls as a whole on gentle 




Fig. 306. — Cross-section of an olive twig at the level of a small tuberch;. 
The open place is a bacterial cavity. Tumor composed chiefly of bark parenchyma, 
the pith and wood cylinder being undisturbed. Result of a pure-culture inocula- 
tion using Bacterium savastanoi. Photographed in 1904. 

shaking. On steamed potato there is often a soluble brownish 
stain (tawny or tawny- white) . This stain also occurs in some 
other media, i.e., water containing peptone and dextrose. 
Potato starch is acted upon a little, the iodine reaction being 
purple while that in the checks is blue. According to Petri, 
potato starch is converted into amylodextrine and maltose. 
The growth, except as influenced by the above-mentioned 
brownish stain, is white on all media. Milk is gradually ren- 
dered translucent (Compare with Nos. IV and XI). Lavender 
or lilac-colored litmus milk becomes blue. No acid is ever 



THE OLIVE tubercle: CAUSE 399 

formed in litmus milk either with or without cream, nor is 
the casein of milk precipitated. The organism has only a very 
slight action on olive oil (Petri, Smith and Brown). Cane- 
sugar is inverted (Petri, Smith and Brown) There is only a 
slight indol reaction. The organism stains readily with Ziehl's 
carbol fuchsin, but not by Gram, It is not acid-fast. It 
grows readily and for a long time in Cohn's solution (very often 
in the form of long rods, sometimes in chains) without fluores- 
cence (Petri says with it) and with the formation of numerous 
crystals of ammonium magnesium phosphate. These crystals 










f^KKKsid^, 






^^ 




Fig. 307. — Section through an olive leaf ^lu)\vins>; structure of a young tubercle 
developing from the lower surface. Result of a needle-prick inoculation. Palisade 
tissue undisturbed. 

become conspicuous in the thin pellicle if the tubes or flasks 
are left undisturbed for a few days. In Cohn's solution with 
1 per cent dextrose, rods in clumped masses occur. In Us- 
chinsky's solution the bacteria are motile and elongated. 
Repeated in 1919: thinly clouded on 4th day; best growth in 
top }y-2 cm.; on 8th day a thin white pellicle, no fluorescence, 
very thinly clouded; motile, short filaments were present; 
after 6 weeks still clouded, not fluorescent. The organism 
grows from 1°C. or below, to 35°C., or a little above. It will 
not grow in bouillon at 38.5°C. and is always killed in -|-15 pep- 
tone-beef bouillon by 10 minutes^ exposure in the water-bath at 



400 



BACTERIAL DISEASES OF PLANTS 



50°C. (10-cc. portions in thin test tubes 17 mm. in diameter 
inoculated from young peptone bouillon cultures). Repeated 
twice in 1920 with same result, following exactly Petri's meth- 
ods. The checks grew promptl3^ The 20 heated tubes re- 
mained clear (20 days). However, 50°C. is not the thermal 
death-point. That is still lower, i.e., between 43°C. and 46°C. 
(Miss Brown); over 45°C. (Miss Elliot). It grows very slowly 
below 5°C. (Petri). 




Fig. 308. — Prof. Luigi Savastano. (Photograph made in Naples at the time he 
was studying olive tubercle.) 

A non-volatile acid is promptly formed from dextrose and 
galactose and this acid appears to be unfavorable to further 
growth. Saccharose is, on the contrary, very favorable to 
growth and less acid reaction is visible when it is used in litmus 
agar. Air is necessary for the production of acid from dextrose 
and galactose, i.e., there is no growth or production of this acid 
in the closed end of such fermentation tubes as yield it in the 
open end. Lactose or maltose added to litmus agar does not 



THE OLIVE tubercle: CAUSE 401 

increase growth, and an alkaline reaction develops the same as 
on plain litmus agar (even in 30 days there is no acid reaction). 
Experiment repeated in 1915 with the same result. Litmus- 
mannit-agar first blues then becomes slowly purple, or red, 
like the litmus-dextrose or litmus- galactose agar. Litmus- 
glycerin agar remains neutral or nearly so for a week or more, 




Fig. 309. — Flagellate rods of Bacterium sarastcmoi EFS, stained by van Ermen . 
gem's silver nitrate method. X 1000. 

and then becomes slowly purple, but never red: repeated 
in 1915 with a good growth of the organism and the same result 
(tubes under observation 67 days). 

Merck's peptone from flesh retards growth (Petri) ; prevents 
all growth (Smith and Brown). Liebig's meat extract retards 
growth (Petri). Beef bouillon is less favorable to growth than 
Witte's peptone in water with saccharose (Petri). A little gas 
is produced in the closed end of fermentation tubes in Uschin- 



402 



BACTERIAL DISEASES OF PLANTS 




Fig 310 —Surface and buried colonies of Bacterium savastanoi on +15 beef- 
peptone agar at end of six days at about 23°C. Pliotographed March 8, 1915. 
X 10. The shghtly irregular outlines are characteristic, j 



THE OLIVE tubercle: CAUSE 403 

sky's solution containing 3 per cent xjdose and the fluid be- 
comes acid (Petri). We could not get this result with our xylose. 
Growth in Winogradsky's solution (nitrogen-free medium) with 
3 per cent glucose, arabinose or xylose, is good, but in the same 
with 3 per cent saccharose, lactose or mannit, is scarcely appre- 
ciable (Petri). We could not verify these statements. A culture 
20 days old in Dunham's solution, when tested for indol, gave 
a reddish color (Petri). Not much indol is produced (Smith, 
Petri). Confii-med by Miss Brown in 1915. Slow-growing, 
white colonies appeared on 1 per cent grape-sugar or cane- 
sugar agar (1.5 per cent agar) reinforced with a neutralized 
decoction of young olive shoots, the tannin compounds inter- 




FiG. 311. — Baclerium savastanoi. Surface colony on +10 beef-peptone gelatin 
37 days at 16°C. Photographed in 1908. X 5. 

fering with development (Petri). No growth for us. It is 
viscid on cooked potato (Petri). The most rapid and abundant 
growth was in Cohn's solution plus 1 per cent anhydrous dex- 
trose, in which there was an abundant white precipitate with 
formation of a thick pellicle and after about 20 days a pale 
greenish color (pea-green) in the upper part of the fluid (Petri). 
Correct, except that we could not verify the greenisTi color with 
the Portofino olive organism (80 days), but did so with the 
Vienna ash organism (28 days and later). Neutral bean agar 
is said to be a good medium for long-continued growth, 
especially when a trace of sodium phosphate or potassium 



404 BACTERIAL DISEASES OF PLANTS 

phosphate has been added (Petri). Petri probably used the 
acid phosphate. Very Hkely there are several strains of this 
organism, all pathogenic, but somewhat different culturally 
(see No. XIV). 

The organism according to my observations does not lose 
virulence quickly on media. It is killed by 30 minutes exposure 
to sunlight in thin-sown agar plates exposed bottom up on ice 
(Fig. 314). Repeated in 1915, exposing for a shorter time, when 
colonies appeared on the insolated half of the plates exposed for 
5 minutes, but none on those exposed for 10 minutes (at 15°C.). 

Technic. — The parasite being abundant and active in cavi- 
ties in the olive tubercle, its isolation offers no special difficulty 
provided young undecayed parts are selected. The surface 
should be scraped and then flamed or soaked 60 seconds to 5 
minutes, depending on size of the piece, in 1 : 1000 mercuric chlo- 
rid water. The piece selected should be crushed thoroughly in 
bouillon, the bacteria being allowed to diffuse for an hour or 
more before plates are poured, which should be both from the 
tube containing the mashings and from two dilutions of the 
same. Success in the first series of plates often depends on 
the care with which the dilutions are made, not only in this in- 
stance but in many others. Various contingencies must be 
provided for, always, especially the two opposite possibilities: 
a, great abundance of viable bacteria in the part selected, which 
would make thick sowings undesirable, or, b, the opposite, which 
would make thick sowings absolutely necessary. 

In making inoculations select rapidly growing soft shoots 
and introduce the bacteria by pricking the shoots several times 
with a delicate needle two inches below the growing-point, 
being careful not to crush or tear the tender bark. Such shoots 
should be selected as will continue to grow for a month or two. 
For comparison inoculate some slow-growing shoots. Leaves 
may be inoculated in the midrib, and in the parenchyma; they 
should be less than half grown, but comparisons may be in- 
stituted by inoculating full grown ones. 

Young streak cultures on slant agar or on potato cylinders 
may be used for the inoculations. 

The writer made one unsuccessful attempt to inoculate 



THE OLIVE tubercle: TECHNIC 



405 




Fig. 312. — Characteristic surface and buried colonies of Bacterium savasianoi 
on +10 beef -peptone gelatin poured November 26, 1910, kept at about 20°C., 
and photographed December 7. X 8 circa. 



406 BACTERIAL DISEASES OF PLANTS 

through stomata, i.e., by spraying young actively growing shoots 
under a bell jar. This should be tried repeatedly (in moist air). 
Is there any special reason why stomatal infections would be 
more than ordinarily difficult? Cut sections of the olive leaf 
and study the stomata. Often, perhaps always, the disease 
begins as a wound infection. 

Olives may be propagated from cuttings, but in most in- 
stances it will be more convenient to buy the few plants needed 
from some nurseryman who makes a business of growing them. 
They should be young plants, and in the extreme southern part 
of the United States, and similar climates, they may be grown 
out of doors, but in most parts of the temperate zones they re- 
quire a warm house. They should be planted in good earth, 
but do not require large space. I grow them preferably in a 
deep bed, but if more convenient they may be in small tubs or 
large pots. Many varieties are subject to the disease. 

If cuttings are made they should be from side branches (ter- 
minal 5 inches), not in new growth. These are bedded (lower 
two-thirds) in sand and watered sparingly, the lower leaves 
being cut away close and the upper ones trimmed back one-half 
to two-thirds. Several months are required to root the cuttings 
and they cannot be used the same year. 

Determine 

For the organism. Morphology. — Size in microns, us- 
ing young cultures on various media. Search for chains, fila- 
ments and pseudozoogloeae. For elongated forms examine 
young cultures in bouillon, Cohn's solution, Uschinsky's solu- 
tion (contrast with Bacterium jnori), peptone water with 1 per 
cent grape sugar, with 2 per cent glycerin, etc. Are capsules 
formed? Petri says they are not. Try viscid potato cultures. 
Search for spores. What are your reasons for thinking none are 
formed? Examine for motility on the margin of a hanging 
drop, using old and young cultures. Stain for flagella, deter- 
mining the number and point of attachment. Select your own 
stain. Is the organism acid-fast? Does it stain by Gram? 
Are there any involution forms? Petri has figured interesting 



THE OLIVE tubercle: MORPHOLOGY 407 

club-shaped and branched bacterial forms found in gastric diver- 
ticula of the larvae of the olive fly (Dacus oleae), and calls them 
Bacterium savastanoi. 

Cultural Characters. — Study and describe the behavior of this 
organism on thin-sown agar plates; in agar stabs and streaks. 
Look for ring-formed colonies in peptone gelatin plates contain- 




FiG. 313. — Suil'ace colony of Bacterium isavaalanui on +10 peptone-beef gelatin 
containing 1 per cent grape sugar. Time, several weeks; temperature 18°C. 
Photographed to show lobes; and the ringed appearance, first mentioned by 
Petri. X 7. 

ing 1 per cent dextrose (Petri). Make very thin-sown +10 
beef-peptone-gelatin plates, keep at 20° to 22°C. and draw the 
peculiar marginal frill. Observe the same marginal behavior 
on gelatin streaks. Savastano likened this growth on gelatin 
to a leaf. Study development of the pellicle in tubes of peptone 
water. Observe the formation of crystals in Cohn's solution, 
e.g., in an undisturbed small flask. Collect them in quantity 



408 BACTERIAL DISEASES OF PLANTS 

from flask cultures, wash free from organic matters and test 
them quahtatively for the presence of magnesium, phosphorus 
and ammonia. 

Describe its behavior in bouillon, and nitrate bouillon. 
Test 10-day and 20-day-old peptone water or peptone-bouillon 
cultures for indol, using Bacillus coll for comparison. Study 
its behavior on potato and other cooked vegetables and make a 
note of any striking appearance not here recorded. Do the 
potato cultures yield a brownish stain? Are they viscid? 

Determine behavior of organism in peptone water in fermen- 
tation tubes with various sugars and alcohols (these fluids should 




Fig. 314. — Agar-poured plate of Bacterium savastanoi insolated 30 minutes 
in April, 1908, and photographed 5 days later. The colony-side was covered 
as far as the line with folds of black paper. 

be titrated and corrected to +15 on Fuller's scale). After a 
week, pipette out the cloudy fluid from the open end of the 
tubes containing dextrose and titrate with phenolphthalein and 
N/20 sodium hydrate; then remove the clear fluid from the 
closed end and titrate as before. Any difference? Try com- 
parative streaks on peptone litmus agar, having previously 
added to some tubes 5 per cent cane-sugar and to others 5 per 
cent dextrose. Any difference in color-reaction or in volume 
of growth? What is the acid formed from grape sugar? 



THE OLIVE tubercle: NON-NUTRITIONAL ENVIRONMENT 409 

Non-nutritional Environment. — Determine sensitiveness of 
the organism to Liebig's meat extract, to concentrated beef 
juice (titrate), to Merck's peptone from flesh, to heat, to freez- 
ing, to sunUght, to dry air, to sodium chlorid (try 2 per cent 
first), to sodium hydrate, and to various acids (citric, maUc, 
tartaric, etc.)- Also test effect of various fungicides. What 
is the optimum temperature for growth? With us it has grown 
better at 20°C. than at 30°C. or above. According to Petri, 
its optimum temperature is about 20°C. Are your experiments 
in conformity with this conclusion? Can you obtain growth on 
or in any medium at 0°C.? Carry on the experiment for several 
weeks and watch your ice thermostat very carefully. Can you 
get it to grow at 37°C.? What is the thermal death-point? 
(See Petri's observation.) 

For the disease. Signs. — After inoculation, how long be- 
fore incipient tubercles are visible on leaves and shoots? Exam- 
ine about every third day. (In my own experiments these 
growths were seldom clearly visible, as such, earlier than the 
end of the second week and the tubercles continued to grow for 
several months.) Describe the gross appearance of the young 
tubercles; of the old ones; of the effect of the disease on roots, 
trunk, branches and leaves. Are the tubercles always fissured? 
Do they contain an unusual amount of tannin, or peroxidase? 

Histology. — With a razor or very sharp knife cut slices free- 
hand of young soft galls (1 to 2 cm. in diameter) and examine 
under the hand lens. Draw some of them enlarged a few diam- 
eters. Examine also at once in water under high powers of the 
microscope in thin, freehand sections. Can you make out the 
bacteria? Can you see them flood out of the tissues? Contrast 
with No. XIV. Fix in Carnoy's fluid, embed in paraffin, and 
cut in various directions to learn the distribution of the bacterial 
cavities and the reaction of the tissues. To what is the water- 
soaked appearance due? Are the bacteria lodged between the 
cells or in their interior? What tissues are chiefly involved in 
the overgrowth? Is there action of the organism at a distance? 
If you have studied crown gall, compare sections of the two 
tumors, and of stems cut between primary and secondary tumors. 
Some of our thin sections have been stained with Ziehl's carbol 



410 BACTERIAL DISEASES OF PLANTS 

fuchsin. This stain gives sharp pictures of the bacteria but is 
apt to overstain portions of the sections. Try amyl Gram, 
which is very good. Can you devise a better single stain, or a 
good double stain? 

Watch young leaves situated immediately above developing 
stem tumors and if internal secondary growths occur along the 
midrib, cut cross-sections of the petiole and of the stem below 
it for presence of the channel of infection leading from the pri- 
mary tumor. If you find it well-developed (as in Fig. 304), i.e., 
as a tiny brown spot, cut also longitudinal sections involving 
its path and make permanent stained preparations. Draw 
some of the bacteria seen in it, together with the surrounding 
parts. 

Is the tubercle corked over? Is there commonly a second- 
ary fungous infestation? What other bacteria have you been 
able to cultivate from natural tubercles? Can you isolate Schiff- 
Georgini's spore-bearing bacillus (Consult Centralb. f. Bakt. 2 
Abt., XV Bd., 1905, p. 198, and U. S. Dept. of Agric, B. P. 
Ind. Bull. 131, Pt. IV, p. 38)'? Berlese's yellow species? 

Variability. — All choice cultivated varieties of olives are 
subject to this disease, and the securing of a profitable resistant 
sort is a work for the future. In Italy the Maremmano and Lec- 
cino are rather resistant (Ferraris, 1915). In California Neva- 
dillo bianco and Manzanillo are more subject to this disease 
than the Mission olive. The greatest variability thus far ob- 
served in this country has been that due to varying degrees of 
cultivation and water-supply. As in pear blight, those infected 
orchards that are well tilled and abundantly irrigated or liable 
to heavy rainfall are most subject to this disease. Frequent 
light rains are also favorable to the spread of the disease, per 
contra dry situations and bright sunshine are unfavorable to it. 

Transmission. — It is generally believed in Italy that wounds 
due to hail-stones favor the entrance of this organism, and also 
those made at harvest-time by the peasants, who thresh the tall 
straggling trees with poles to dislodge the olives. Observation 
shows that the tubercles often originate in scars where leaves or 
branches have been torn off. The reason for these various 
wound-infections was unknown until Home, Parker and Daines 



THE OLIVE tubercle: TRANSMISSION 411 

in California showed that under the action of rain the tubercles 
ooze bacteria freely to their surface from whence they are washed 
to other parts of the tree. They conclude that: "The infected 
trees must be covered during the rainy weather of winter with 
an almost continuous coating of the specific bacteria." Prob- 
ably birds and insects also help to spread the disease, but exact 
experiments, so far as I know, are wanting. Petri in Italy has 
shown, however, if not absolutely at least with a fair degree of 
conclusiveness, that the olive fly, Dacus oleae, carries this organ- 
ism along with the yellow bacillus (Ascohacteriwm luteum Babes) 
in its salivary gland and intestinal diverticula as a regular 
(symbiotic) occupant (vide Centralb. f. Bakt. 2 Abt., XXVI 
Bd., p. 357, and more especially "Ricerche sopra i batteri intes- 
tinali della Moscaolearia," Memorie d. r. staz. di patologia vege- 
tale, Roma, 1909, from which also I have taken various accredited 
citations under Cultural Characters, etc.). The second organ- 
ism mentioned is the yellow saprophyte so often found in the 
olive tubercle. Also as in pear blight, it is likely that the dis- 
ease is sometimes spread by pruning instruments, and certainly 
it must be brought into the orchard frequently from the nursery. 
In California during the dry summer season no new infections 
occur but with the coming on of winter rains and of spring rains 
infections are numerous. Home and his colleagues believe that 
infections take place without insect wounds, by growth of the 
bacteria in bark crevices from which they penetrate the deeper 
tissues especially over places subject to tension from rapid 
internal growth, and that varieties with a smooth bark and an 
open habit of growth like the Mission olive are, for these reasons, 
freer from the disease than those having a rough bark and a more 
compact habit of growth. 

literature 

Read: (1) Smith, Erwin F., "The Olive Tubercle," Science, 
N.S., Vol. XIX, p. 416, March 11, 1904; (2) Do. " Some Observa- 
tions on the Biology of the Olive-Tubercle Organism," Ce7i- 
tralblatt fur Bakteriologie, etc., 2 Abt., XV Bd., 1905, p. 198; 
(3) Do. "Recent Studies of the Olive-Tubercle Organism," 
Bull. 131, pt. IV, Bureau of Plant Industry, U. S. Dept. Agri- 



41-2 BACTERIAL DISEASES OF PLANTS 

culture, Washington, Govt. Printing Office, 1908; (4) Home, 
Parker, and Daines, "The Method of Spreading of the Olive- 
Knot Disease," Phytopathology, Vol. II, June, 1912, p. 101; 
and (5) Home, W. T., ''The Olive Knot," Monthly Bulletin, 
State Commission of Horticulture, Sacramento, California, 
August, 1912, p. 592. 

Consult also ''Bacteria in Relation to Plant Diseases," Vol. 
I, 1905, Figs. 38 and 57, and Plate 2; and Vol. II, Plates 6 and 9, 
and Fig. 23. Other recent literature is mentioned in Bulletin 
131 (above reference number 3), where the name Bacterium 
savastanoi was first used. 

The book by T. Ferraris is "I Parassiti vegetale delle piante 
coltivate od utile," Hoepli, Milano, 1915. 



XIV. THE CROWN GALL 

(Syn. Plant Cancer) 

Type. — This also is an overgrowth, but of a different kind 
from the preceding. Crown gall is a disease of wide geograph- 
ical distribution, occurring on a great variety of cultivated 
plants (Figs. 315 to 319) and on some wild ones, e.g., the chest- 
nut. It is primarily a disease of the parenchyma but it is unlike 
any of the diseases hitherto described in that the cells of the at- 




FiG. 315. — Crown gall on hop. Paris daisy strain. A pure-culture inoculation. 
Time, about 3 months. April, 1907. 3'3 nat. size. 

tacked parts are not disintegrated and killed, but on the contrary 
are induced to multiply, the result being an imperfectly vas- 
cularized, covered or naked, irregular, soft or hard overgrowth, 
or tumor, composed in part at least of masses of rapidly dividing, 
round or spindle-shaped cells of reduced size, forming a hyper- 
plasia, which on some plants under favorable conditions may be 
larger than the root or shoot that bears it (Fig. 320) but then 

413 



414 



BACTERIAL DISEASES OF PLANTS 



frequently decays readily, especially on soft plants like the sugar 
beet and the willow (Figs. 321, 322). The nature of the very 
large hard tumors on oak trees common in the United States is 
still undetermined. The largest crown-gall I have ever seen 
weighed 96 pounds; this was received in 1919 by Dr. B. T. Gal- 
loway and is now in our collections. It was found on the stem 




Fig. 316. — Dwarfing effect of crown gall on sugar-beet. Pure-culture in- 
oculation. The organism used was plated from a gall on Paris daisy. Tumor 
larger than the root. Time, 37 days. Inoculated June 3, 1907. 1-2 nat. size. 

of a wild fig on an island in. the Florida everglades 40 miles 
southwest of Miami (Florida State Park). Dr. I. B. Pole 



3-^ nat. size. A serious disease of roses. (3) On apple limb. Above-ground 
tumors — one growing out of pruned end. From an apple tree in North Carolina. 
1911. ^^ nat. size. Often the entire tree is attacked in this manner. (4) 
On a pear seedling grafted by Hedgcock with fragments of a rose-gall in the sum- 
mer of 1907. Photographed December 23, 1907. ^^ nat. size. See next page. 



THE CROWN gall: TYPE 



415 




Fig. 317. — Crown galls due to Bacterium timiefaciens: (1) On hop. Natural 
infection from Washington State. 1908. M nat. size. Stem dwarfed above the 
tumor. (2) On rose. Natural infection from a New Jersej^ rose house, 1909, 



416 



BACTERIAL DISEASES OF PLANTS 




Che!r,f!!f ~.^'''', r''*r ^"°^"l^ti«"'^ «f ^••o^™ gall: (1) On yellow Paris daisy. 
Check stem at nght. Tnne, one month. February, 1907. The upper softer 



THE CROWN gall: TYPE 417 

Evans, of Pretoria, has reported still larger ones from the 
willow in South Africa (20 inches long with a circumference of 
4 feet, 7 inches). 

This disease is of a peculiar type: (1) in that the growth 
is extra-physiological and injurious to the rest of the plant, 
slowly dwarfing or killing it; (2) in that secondary tumors occur 
as growths from tumor-strands which are bedded deep in the 
normal tissues (Figs. 319, subs. 5, 6, and 323 to 325) and derived 
by growth (cell-division), in the form of a continuous chain of 
cells, from the primary tumor; and, finally, (3) in that the 
secondary tumors reproduce the structure of the tissues in which 
the primary tumor has developed even when they appear in other 
organs, thus if the primary growth is in the stem and the second- 
ary is in a leaf, the attacked part of the leaf will be converted 
into a pseudo-stem (Figs. 326 and 327, sub. 4). As bone is 
often out of place in malignant animal tumors, so lignin may be 
out of place in crown gall (Fig. 328). 

The largest tumors are developed out of the cambium, but 
small ones may be produced by very shallow punctures into the 
bark parenchyma and these become vascularized (Fig. 329). 
I have not always been able to produce galls by inoculations 
into the pith. Much depends on its age. The growth of the 
tumor may stimulate multiplication of the surrounding uninocu- 
lated tissues. For evidence of this, see the lower part of Figure 
329 (under X) where large bark-parenchyma cells are subdivid- 
ing, and (Fig. 319, subs. 5 and 6) where the wood is greatly 
thickened on the side of the stem bearing the tumor strand. 

Attacked branches are frequently killed (Figs. 330 and 319, 
sub. 3), but seldom is the whole plant killed, at least not for a 
long time, if it is of any size when attacked. Attacked plants 
are generally more or less stunted in their growth, especially if 



part of the shoot has developed the larger tumors, (la) On yellow Paris daisy, 
showing tumor due to a single infected needle prick. Sterile pricks above. (2) 
On peach. Time, 5 months, nearly. The organism used was plated from a 
gall on the peach. 1908. (2a) On peach. Time, 18 days. (3) On apple. 
Inoculation from peach, made high up on the stem by needle pricks. 1908. 
% natural size. Time, 5 months. Stem above is dwarfed. (4) On European 
grape. With bacteria plated from crown gall on poplar. Time, 44 days. 1910. 
A serious disease on raisin grapes (Muscats) in California. 

27 



418 



BACTERIAL DISEASES OF PLANTS 




ax 



Fig. 319. — Pure culture inoculations of Bacterium tumefaciens: (1) On Jap- 
anese radish. The organism used was plated from a radish. 1912. y^ nat. size. 



THE CROWN gall: TYPE 419 

the tumor is centrally located and then the plant may be killed 
outright, but this is much less frequently the case than with 
animals attacked by cancers, the anatomy and physiology of the 
plant being specially unlike that of animals in that there is no 
central digestive, nervous, or circulatory system subject to 
attack, with a disastrous reaction upon the whole organism. 
The nearest analogy to this is the growing point of centrally 
growing soft plants like young sugar-beets. If the needle is set 
in so that this is interfered with, as the big tumor develops, death 
occurs within a few weeks. See Jour. Cancer Research, Vol. I, 
No. 2, PI. Vila — All of the inoculated plants there shown died a 
few weeks later. I have obtained the same results on tobacco. 

The organism causing this tumor occurs only inside of cer- 
tain of the proliferating cells. When the cell divides, the or- 
ganism is carried over into the daughter cells, in at least a part 
of which it multiplies. It occurs in the cell in comparatively 
small numbers and, owing to the granular nature of the proto- 
plasm cannot be made out satisfactorily even with high powers 
of the microscope. By means of gold chlorid impregnation 
followed by formic acid we obtained a deep blue-black stain 
in certain rod-shaped bodies in the tumor cells (Figs. 331, 332) 
and for a time these were interpreted as the intra-cellular bac- 
teria, but I now regard them as mitochondria. 

In 1916 the writer discovered crown galls bearing leafy shoots 
and subsequently produced many by needle-puncture inocula- 

(2) On Paris daisy. 191L Time, 73 days. Primary tumor at A^ on stem, 
secondary tumors in 3 leaves. These were connected with the stem-tumor by 
tumor-strands lying deep in the wood. 3*2 nat. size. (3) On Paris daisy. The 
original tumors (X, X) have decayed and a new tumor has grown out below. 
Stem dead. Time, 10 months, nearly. % nat. size. (4) Sunflower. Inoculated 
in the disk when young (August 14, 1915) with isolation from the hop (4-day 
agar-streak). A tumor-strand passed through the pith rupturing to the surface 
below in two places — that shown and as a small tumor in axil of Y. \^ nat. size. 
(5) On Paris daisy. Cross-section of stem showing a primary tumor below and 
a large green tumor-strand with thickening of wood on that side. The strand 
was under strong pressure and protruded when the stem was cut. X 2. (6) 
On Paris daisy. Inoculation made on stem below cut here shown. Observe 
one-sided thickening of wood, and three tumor -strands which passed to as many 
leaves, two fusing. The leaves, which bore secondary tumors, were cut away 
some weeks earlier and from the stubs new tumors have grown out. Time, March, 
1911. 



420 



BACTERIAL DISEASES OF PLANTS 




Fig. 320. — Crown gall on sugar-beet due to Bacterium tumefaciens. A pure- 
culture inoculation of 1913. The organism used was plated from a hop tumor. 



THE CROWN gall: TYPE 421 

tion on stems and leaves of tobacco, Pelargonium and other 
plants. 

Earlier than this by some years, Miss Brown and myself had 
demonstrated that crown galls may bear roots (hair}' root of 
apple, etc.) but I did not then perceive the full meaning and 
trend of this discovery, viz., that because the type of a crown gall 
depends on the kind of tissues inoculated it should be just as 
easy to produce tumors bearing leafy shoots or flower buds as 
roots. This had to be stumbled upon to be seen, like many 
another perfectly obvious thing. Its discovery, however, it 
seems to me, adds very considerably to our knowledge of the 
nature of crown gall and throws a flood of light also on the origin 
of animal teratomas. 

The subject of teratomas is so interesting that I have included 
a number of our more striking results (Figs. 333 to 344 and 347, 
!348). 

The common name ''crown gall" serves to recall the fact 
that the galls are found very often on the trunks of various fruit 
trees at the surface of the earth, i.e., on the part known to 
gardeners as the "crown" of the plant. They may occur, how- 
ever, on any part of the root or shoot, being very common above 
ground on the branches of the daisy, grape, quince, apple, rose, 
willow and poplar. They are also common on the roots of a 
variety of plants but must not be confused with root galls due 
to nematodes. 

This disease is common in many localities in North America, 
Europe, South Africa (Fig. 322), and other parts of the world, 
and is coming to be recognized in the United States as a more 
serious disease than it was formerly supposed to be. 

Cause. — Crown gall is due to Bacterium tumefaciens Smith 
and Townsend. This is a small, white, motile, polar flagellate 
(Fig. 349), non-sporiferous, Gram negative, non-acid fast, non- 
liquefying, non-nitrate-reducing, aerobic, non-gas-forming, non- 
starch-destroying, dry-air-sensitive, sunlight-sensitive, chloro- 



The inoculation was by needle pricks from a 1-day agar-streak culture. Leaves 
cut away to get a clearer view. Actual size (longest way) of the tumor a little 
more than 5 inches. Time, 3 months. Tumor larger than the root and still 
sound, i.e., free from necrosis. 



422 



BACTERIAL DISEASES OF PLANTS 




Fig. 321.— Crown gall on sugar-beets due to Bacterium tumefaciens. Pure- 
culture, needle-prick inoculations, using organism isolated from a tumor on hop. 
Time, 104 days. Necrosis has begun. Tumors larger than the roots. Actual 
diameter about 4 inches. 



THE CROWN gall: CAUSE 



423 




Fig. 322. — Crown galls on willow due to Bacterium tumefaciens. Natural 
infections from South Africa received in 1911. Frequently these willow galls are a 
foot or more in diameter, and at a distance the attacked trees look as if large birds 
were roosting in them. % nat. size. 



424 



BACTEEIAL DISEASES OF PLANTS 




I Fig. 323. — Tumor-strand of crown gall in Paris dai.sv . The result of a pure- 
culture inoculation. Cross-section of stem between a primary stem-tumor 
(caused by needle pricks introducing Bacterium tumefaciens) and a secondary 
tumor in a leaf. It shows a tumor-strand in the inner wood and beyond it 
normal wood (above) and normal pith (below). 



THE CROWN gall: CAUSE 



425 




Fig. 324. — >Sho\ving crown gall on Pans daisy, results of pure-rulime uiocula- 
tions: (1) Radial longitudinal section through normal bundle of petiole for com- 



426 BACTERIAL DISEASES OF PLANTS 

form-tolerant, sodium chlorid-tolerant (up to 3.5 per cent), 
lab-producing, acid-forming (with certain sugars) rod-shaped 
schizomycete, which grows on agar-poured plates in the form of 
small circular, somewhat raised, wet-shining, translucent colo- 
nies. Streaked on agar from agar the growth is smooth and 
shining, but when streaked on agar from peptone bouillon the 
various strains (daisy, hop, etc.) usually give a thin, wrinkled 
and dull surface. On gelatin plates the surface colonies are 
circular, small, dense, white and non-liquefying. In -|-15 pep- 
tone bouillon after some days there is more or less pellicle and a 
whitish rim of stringing, gelatinous threads ; earlier (48 hours) the 
bouillon contains numerous delicate suspended filaments which 
are best seen with oblique light and on shaking. In old undis- 
turbed bouillon cultures there is a rather firm whitish pel- 
licle and very little clouding of the fluid. There is a white 
transient growth on steamed potato with some graying of the 
substratum. Growth in Uschinsky's solution is scanty; in 
Cohn's solution, scanty or absent. In milk the casein is thrown 
out of solution but only after several days. Litmus milk is 
blued (never reddened) and the litmus is frequently reduced. 
The living organism takes up Congo Red from culture media 
containing it and in this way may be distinguished from the 
root-nodule organism of legumes (Karl Kellerman). Verified in 
1919. Indol production, scanty. There is an invertase, and a 
lab ferment. Some ammonia and hydrogen sulphide are pro- 
duced. It grows from 0°C. to + 37°C. The optimum tempera- 
ture for growth lies between 25° and 30°C. The thermal death- 
point (10 minutes exposure in the water-bath in test tubes in 
+ 15 peptone beef bouillon) is approximately 51°C. It shows 
slight toleration for organic acids (malic, citric, acetic) and still 

parisoij with 2. Spiral vessels at the left, pitted vessels at the right. (2) Longi- 
tudinal section through a petiole showing tumor-strand in a bundle. Spiral 
vessels at the left, pitted vessels at the right. (3) Cross-section of stem between 
primary and secondary tumors, showing large-celled tumor-strand with big 
nuclei. Pith below, inner wood above. Tumor-cells wedging apart the spiral 
vessels. (4) Cross-section of a stem between tumors. Pith below, wood above; 
in the center is a tumor-strand developing tracheids out of certain of its cells. 
They contain nuclei and are still immature. The vessels above are the normal 
spiral vessels of the inner wood. 



THE CROWN gall: CAUSE 



427 




Fig. 325. — Crown-gall tumor-strand in coarse-celled cortical parenchyma of 
a Pelargonium teratoma. Diffuse tumor tissue with tracheids at the right and 
lower left. From a pure-culture inoculation. Slide stained by Lucia McCuUoch. 
Photomicrographed by the writer. 



428 BACTERIAL DISEASES OF PLANTS 

less for sodium hydrate. There is more growth in +15 than in 
— 15 peptone bouillon. The optimum acidity for this organism 
in beef bouillon appears to lie between +12 and +24 on Fuller's 
scale (1.2 to 2.4 per cent of N/1 acid). 

The bacterium is sensitive to germicides and slowly loses 
virulence on culture media. It passes over easily (under action 
of cold, sodium chlorid, or acids) into club-shaped, Y-shaped, 



t: ;._^^ 



Fig. 326. — Crown gall on Paris daisy. Cross-section of a secondary tumor 
in a petiole, the result of a pure-culture inoculation on the stem. This has rup- 
tured to the surface at the top and left side with destruction of the normal tissues. 
In its center is a tumor-strand connecting it back to the primary tumor. It 
shows crudely the structure of a stem (wood, cambium, phloem). In the lower 
part of the tumor are vessels running at right angles to the longer axis of the stem. 

and variously branched involution forms (Fig, 350) which often 
are dying or dead, i.e., will not grow on agar-poured plates, or 
come up slowly. These moribund involution forms occur not 
only in culture media but are common in the tumor, and to them 
must be attributed not only our former difficulty in isolating the 
organism, but also the failure of others to isolate it. To keep 
the organism alive on ordinary culture media, store the cultures 



THE CROWN gall: CAUSE 




Fig. 327. — Early stages of crown gall in Paris daisy due to Bacterium tume- 
faciens. (1) Stem 10 days after inoculation by needle-pricks. Tumor just 



430 BACTERIAL DISEASES OF PLANTS 

in a cool box and make frequent transfers (once every 3 weeks). 
It lives longer in milk and bouillon than in agar. 

Bacterium tu7nefaciens is cross-inoculable on a great variety 
of plants, e.g., using strains cultivated from the daisy and hop 
we have produced galls on more than 40 kinds of plants belong- 




FiG. 328. — Crown gall on Paris daisy. Result of a pure-culture inoculation. 
Section of a tumor showing lignin deposited out of place, i.e., on the walls of 
three cells of the large-celled petiole parenchyma (Cortex). Stained with methyl 
green and acid fuchsin. 

ing to 18 families, as follows : daisy (2 sp.) tomato, potato, tobacco 
(3 sps.), oleander, cabbage, cauliflower, turnip, radish, beet, car- 
rot, grape, clover, peach, almond, raspberry, apple, pear, carna- 
tion, hop, Coleus, Citrus, Impatiens (2 sps.), Opuntia, Persea 

beginning ; epidermis pushed up. (2) Longitudinal section through a young un- 
ruptured secondary tumor in a petiole. Normal tissue under pressure at either 
side and bulging. Result of a pure-culture inoculation. (3) A part of 2 enlarged. 
The round spots are nuclei in the cells of the tumor. The bacteria are invisible. 
(4) Cross-section of a petiole showing the central leaf-trace converted into a second- 
ary tumor which has not yet ruptured : tumor-strand in the center, bej^ond which 
is a whorl of wood-wedges not well lignified (the lignified parts are stained dark), 
beyond these cambium and then phloem. Beyond the tumor is the large-celled 
leaf parenchyma, which is compressed and bulging. Result of a pure-culture 
inoculation on the stem. Normal leaf-traces above and below. 



THE CROWN gall: CAUSE 



431 




Fig. 329. — Crown gall confined to the bark parenchyma of a Paris daisy 
stem but containing tracheids (above X), induced by needle inoculations }i mm. 
deep. Under and at the left of X there is action at a distance, i.e., large cells 
like X are dividing as if by a stimulus received from the neighboring hyperplasia. 
The same phenomenon has been observed in crown gall on tobacco. (Consult 
Jour. Cancer Research, Vol. I, No. 2, Plate I, Fig. 3, and Plate XXIII, Fig. 78.) 



432 



BACTERIAL DISEASES OF PLANTS 



(with difficulty), Juglans, poplar, Pterocarya, AUemanda, mango, 
stock, Ricinus, cassava, Fuchsia, Reseda, Salvia, Pisum, Calen- 
dula, Helianthus, etc. 

Technic. — The writer and his associates experienced great 
difficulty in first isolating this organism from crown galls but 
now that the obstacles are known they are easy to evercome and 
any one with ordinary technical ability can demonstrate the 



'4 



^.^^ 



^ 





Fig. 330. — Crown gall on white Paris daisy (Chnjsanthemum frutescens). 
Plant inoculated December 13, 1906, with a pure culture of Bacteriuvi tumefaciens 
(plated from a tumor on the daisy) and photographed 7 months later. One branch 
killed, j'i natural size, circa. 

occurrence of the parasite in sound galls by the poured-plate 
method, with exception of certain problematic galls occurring 
on the sugar beet. The organism is best isolated from young and 
rapidly growing tumors, from which it may sometimes be had in 
practically pure culture. Old galls are apt to be filled with white 
and variously colored non-parasitic schizomycetes, especially 
with yellow and white ones, and may contain mites, nematodes, 
yeasts, myxomycetes, and various fungi. 



THE CROWN gall: TECHNIC 



433 



Owing to the fact that the parasitic organism occurs in the 
tissues in comparatively small numbers (very small numbers as 
compared with tissues subject to any of the diseases previously 
mentioned), and further to the fact that a large proportion of 
such as do occur are in a dormant condition, considerable quan- 
tities of the tumor should be taken for the cultures, and on a 
portion at least of the plates the inoculations should be very 




• 




# 







•r 



Fig. 33L — Crown gall on Paris daisy showing rod-shaped, mitochondrial 
(?) bodies photographed in place within the cells. Thin section of a portion of 
ten tumor cells after treatment with gold chlorid and formic acid and counter- 
staining with eosin for the nuclei. X 1000. 

heav\^ (6 to 8 three-millimeter loops) ; moreover, the plates 
should not be discarded until the end of the third week. Those 
colonies which come up on the poured plates from the 4th to the 
8th daj^ or later are more likely to be the organism sought than 
those which appear during the first three days. The plates 
may be kept at 20° to 30°C. 

28 



434 



BACTERIAL DISEASES OF PLANTS 



A very good procedure is to flame lightly and pare away 
the exterior of the tumor with sterile knives, then remove some 
of the sound-looking interior, plunge for 3 seconds into mercuric 
chlorid water (1 :1000), then wash for about the same time in 
distilled water, and crush it thoroughly in a little sterile water 
or bouillon, using a cold sterile knife -blade on the bottom of 
a sterile Petri dish (or if great care is taken it may be done 




Fig. 332. — Thin sections of crown gall of the daisy. Tissues stained by means 
of gold chlorid. Nuclei out of focus are visible in each photograph, and at X 
there is a Y-shaped rod. 

inside a thick- walled tube in bouillon), using considerable force. 
The cloudy fluid in the dish should now be pipetted and the 
mashings scraped and poured into a tube of peptone water, 
beef bouillon, or autoclaved water, and allowed to stand for 

has ruptured through to the surface. Stem inoculated by needle-pricks in two 
leaf axils using Bacterium tuniefaciens plated from a tumor on hop. Inoculated 
September 29, 1916. Photographed November 24, 1916. Nat. size. (See next 
page.) 



THE CROWN gall: TECHNIC 



435 




Fig. 333. — .4. Inoculated crown-gall teratoma on cauliflower. The pale 
shoot above the naked tumor has a tumefied sarcomatous interior. X 5 circa. 
B. Same as A, but a side view. At A' the sarcomatous tissue in the shoot 



436 



BACTEEIAL DISEASES OF PLANTS 




\ 



Fig 334 —Crown-gall teratoma on Ricinus communis (castor oil plant). 
Inoculated with hop strain of Bacterium tumefaciens in two places by needle- 
pricks March 25, 1916. Photographed April 29, 1916. ^f nat. size. Leaves 
reflexed and dying. Secondary tumors on petiole at X. The tumor-bearing plant 
was of the same age and size as the check when it was inoculated. 



THE CROWN gall: TECHNIC 



437 




Fig 335 —Crown gall on common tobacc-.., iK'anng leaves and flower-buds. 
Leaves twisted, fasciated and tumefied at their base. Inoculated by the writer 



438 BACTERIAL DISEASES OF PLANTS 

some hours to diffuse. Then make the plates, pouring some 
directly from the tube containing the mashings and others 
from dilutions of it (several drops of the cloudy fluid into the 
second tube of bouillon, and after it has stood for an hour, with 
some shaking, ^2 cc. from this tube into a third tube of bouillon). 
Rarely does one obtain colonies on plates poured from the 
dilutions when first made. Moreover, if your material is scanty 
you will of course save not only the remains of the tumor but 
also your original tube and the dilutions made therefrom, and 
pour another series of plates next day, but if there is a green 
stain or if gas is forming in the tube containing the mashings, 
then pour only from the dilutions. For these poured plates 
use +15 peptone-beef agar. 

On the poured plates all yellowish, orange, greenish, or 
pinkish colonies, all branching white colonies, and all circular 
white colonies, if opaque, are negligible. Only those colonies 
that come up slowly, that remain for a considerable time small, 
circular, raised and glistening-translucent (watery) need be 
considered (Figs. 351, 352). Even following this advice some 
of the colonies selected for the sub-cultures (which may be on 
agar or potato or in bouillon) may not prove to be infectious, 
therefore it is advised to experiment with quite a number of 
colonies, and to examine them by transmitted light, rejecting 
all that show a narrow clear zone about the colonies even if 
they look right by reflected light. 

For inoculation purposes the student has choice of many 
kinds of plants since many are susceptible. For class work the 
young and rapidly growing roots of turnips or sugar-beets, the 
soft shoots of tomatoes, Pelargoniums, castor oil plants, or 
Paris daisies, and the crowns of young peach, almond, or poplar 
are recommended. Shoots and crowns of the hop or the 
European grape, if growing satisfactorily, may also be used; 



July 7, 1916, on the cut surface of the middle of an internode of the main axis 
near the top of the plant just before blossoming time, by needle pricks, using a 
pure culture of the hop strain of Bacterium tumefaciens, which had been passed 
through sunflower. Photographed August 4, 1916. Natural size. There were 
11 flower -buds on this date but a day or two later they began to fall off without 
opening. The big side branches developed after the inoculation. 



THE CROWN gall: TECHNIC 



439 




Fig. 336. — Crown galls on tobacco. A. Teratoma containing perhaps 100 
leafy shoots. Inoculated July 29, 1916, by needle pricks on the cut end of the 
stem (middle of an internode) introducing the hop strain of Bacterium tumefaciens 
from a 48-hour agar culture. Photographed September 1, 1916. ^i natural size. 

B. Like A but inoculated ]March 1, 1918 (from hop through sunflower) on a 
cut internode of the main axis. The white part of the tumor was very smooth, 
free from chloroplasts and covered by an epidermis, i.e., the malignant part lay 
deeper. Twelve of 13 inoculated internodes contracted the disease. Photo- 
graphed May 8, 1918, natural size, nearlj-. 



440 



BACTERIAL DISEASES OF PLANTS 




^ 












Fig. 337. 



THE CROWN gall: technic 441 



Fig. 337. — Crown-gall witch l)room on the cultivated carnation dvie to 
Bacterium fumefaciens (a natural infection). There was no marked tumefaction 
at the base of the shoots but, as the sections showed twisted tissues, plates were 
poured and white colonies were obtained. Six of these were sub-cultured and 
tested by the writer on Ricinus and tobacco. Colonies 1, 3, 4, 5 and 6 failed 
to produce any growths, but colony 2 gave sarcomatous tumors on both plants 
and also on carnation. Photographed November 11, 1916, ^<7 nat. size. 



442 



BACTERIAL DISEASES OF PLANTS 








^'*- 



Fig. ooS. — Ciown-gall teratoma on Pelargonium. From a pure-culture 
inoculation of Bacterium tumefaciens made by the writer. Photographed January 
11, 1916. X 4. Time 12 weeks. 



THE CROWN gall: TECHNIC 



443 





Fig. 339. — A. Section of crown gall on tobacco due to Bacterium tumefaciens. 
Teratoma developing on the cut end of an inoculated internode. Tumor below 
and a dwarfed shoot above developing out of it. 

B. Tissue of leaf from A (at A') showing the pahsade tissue reversed, i.e., 
facing awav from the sky. 



444 



BACTERIAL DISEASES OF PLANTS 




Fig. 340. 



THE CROWN gall: technic 445 



Fig. 340. — A. Crown-gall teratoma produced by needle pricks on cut inter- 
node of Nicotiana tabacum using hop strain of Bacterium himefaciens passed through 
sunflower in 1915. Photograph shows fused tumefied leaves, and sprouts growing 
out of all parts of them. At A', X, X, .shoots 3 and 4 inches long were cut away. 
Inoculated March 1, 1918, with 14-day agar-streak culture. Photographed May 
8, 1918. Natural size. 

B. Same phenomena as in A, but on a petiole of Nicotiana sylvestris. Hop, 
colony 1, check on flask N. Inoculated January 16, 1917. Photographed March 
2, 1917. X 2. There is a continuous tumor on the margin of the petiole with 
leafy sprouts in five places (A' — X). 



446 BACTERIAL DISEASES OF PLANTS 



Fig. 341. — A crown-gall teratoma on common tobacco showing an abnormal 
organ, shoot (?), leaf (?). Probably a stem, because its vascular system comes off 
the normal vascular cylinder and is itself an irregular cylinder. This growth 
is a blunt, cylindrical, curved, horn-like, white body, pale greenish at the swollen 
base and bearing 20 abortive green or greenish leafy organs (shoots), most of which 
are borne on longitudinal seams as if on rudiments of decurrent leaf wings. Pos- 
sibly it is a modified leaf as it does not arise in any leaf axil. The largest and green- 
est of the leafy outgrowths are 3 at X along a seam which extends to the top of the 
horn. Stem inoculated in the leaf axils November 26, 1916, with sub-culture 
of colony 2 {Bacterium tumefaciens) from carnation witch broom (see Fig. 337). 
Photographed January 24, 1917. X 4. 



THE CROWN gall: TECHNIC 



447 




Fig. 34L 



448 



BACTERIAL DISEASES OF PLANTS 




Fig. 342. 



THE CROWN gall: technic 449 

likewise, soft shoots of young tobacco or the undeveloped disks 
of the sunflower. For study of the leafy tumors (teratomas), 
inoculations may be made in the leaf axils or on the cut inter- 
nodes of various plants. The writer has used chiefly tobaccos 
and the common hothouse Pelargonium. In the same way, 
for tumors containing roots, the tops of Impatiens halsamina 
(the common garden balsam) ma}^ be inoculated. For study 
of the tumor-strand and secondary tumors, the young rapidly 
growing shoots of the Paris daisy [Chrysanthemum frutescens) 
are best, and the inoculations should be made toward the top 
of succulent stems which should continue to grow vigorously 
for at least 2 months. The needle should be thrust into the 
stems immediately under the leaves. Peklo in Bohemia 
(1915, 1. c.) obtained very good tumor-strands in the stem of 
the sunflower by inoculating into the young flower disk, and the 
writer has verified his statements. Hop does not infect daisy. 

The result of the inoculations will be successful and inter- 
esting in proportion to the virulence of the organism and the 
activity of the plant. Well nourished rapid-growing plants 
yield much larger tumors than slow-growing ones. To demon- 
strate killing effects of the gall use young sugar beets or young 
Nicotiana sylvestris, inoculating in the center of the crowm. 

Cuttings of the Paris daisy, if made in the hothouse in 
September, should give plants suitable for inoculation in 
November and December. The slips should be end branches 



Fig. 342. — Crown-gall teratoma on orange due to Bacterium tumefadens. 
Stem inoculated January 14, 1916. This is my No. 48 (hop strain through sun- 
flower, colony 1). It was made by needle pricks in the region of a dormant bud, 
but nothing in the way of a tumor developed either in 1916 or 1917. I looked 
at it many times. Other inoculated orange buds developed slight tumors bearing 
supernumerary buds and then died. The small firm dark green shoot marked A' 
is an outgrowth of the stem. This appeared in 1917 but with no evidence of 
any tumor at its base. Three other shoots and about 12 buds developed from the 
tumor itself and all of them were soft and light green. This young, rapidly growing 
tumor appeared within 3 or 4 weeks of the time the photograph was made. The 
tumor is interesting as having remained dormant for two years and then begun 
to grow rapidly as an embryoma. The rough white part of the tumor is the 
naked sarcoma. Photographed March 2, 1918. • X 3. Sections of leaf C and of 
shoot y between a and b were cut and examined for a tumor -strand but none was 
found. 

29 



450 



BACTERIAL DISEASES OF PLANTS 




tiu. 343. 



THE CROWN gall: technic 451 

not in flower-bud or in blossom. They should be set into 
shallow boxes in sand in close rows, and the foliage trimmed 
up considerably. Here they should remain for about 3 weeks, 
i.e., until a callus has formed and roots begin to push. Bottom 
heat is not necessary, as they root readily. They should 
then be put into good soil in thumb pots and transferred from 
time to time (rather frequently) to larger pots so as to keep 
them growing rapidly. There should be no check whatever 
in their growth else they will bloom prematurely. Pelargonium 
slips should be treated in the same way. Peaches and almonds 
should be planted in similar boxes of sand after carefully cracking 
and removing the shell, without injury to the kernel. Hard- 
shell almonds as they come upon our markets are more likely 
to germinate than the bleached thin-shell almonds. Two or 
three months must be allowed for growth. Ricinus, tomatoes, 
tobacco, sunflowers, sugar-beets, and turnips should be grown 
from seed. Allow at least 2 months. Cuttings of willows and 
poplars may be rooted in the spring for use in houses the follow- 
ing winter or spring. 

Determine 

For the organism. Morphology. — In various media, size 
in microns; form; aggregation of elements, i.e., chains, filaments, 
pseudozooglcese; motility, presence and distribution of flagella 
(use Pitfield's stain); absence of endospores; capsule stain; 
Gram's stain; acid-fast stain; character of the involution 
forms. 

Cultural Characters. — Size and appearance of colonies on 
thin-sown agar and gelatin plates; stabs and streaks on agar; 
ditto on gelatin; behavior in peptone bouillon (watch early 

Fig. 343. — Hard crown-gall embryoma, on mango {Mangifera indica). Ter- 
minal bud inoculated by the writer January 19, 1916, by needle pricks using the 
hop strain of Bacterium tumefaciens (sunflower Colony 1). This tumor contains 
6 distinct centers of embryonic growth. For one of the larger ones (under the 
arrow) see Fig. 344A. Surface brown except the embryonic parts which were green. 
The leaves surrounded by the tumor, and appearing to grow out of it, are 
stem leaves. Time, 20 months (nearly). Photographed by James F. Brewer, 
September 11, 1917. ^4 nat. size. Actual size of the tumor 63^^ by 43'2 hy 4 
inches. 



BACTERIAL DISEASES OF PLANTS 




Fig. 344. 



THE CROWN gall: CULTURAL CHARACTERS 453 



Fig. 344. — A. Slow-growing crown-gall embryonia on mango. In the center 
(under X) a folded (twisted) green bud. Notice also a bud at the extreme left, 
one in the right upper corner, one below it, and one in the center under the main 
growth. These five I have counted as one of the six centers of embryonic growth. 
See Fig. 343. This embryonic portion was under observation many weeks but 
it developed no shoots. X 4.5. 

B. Cross-section of a very young orange fruit showing crown-gall tumors in 
the placental region. Bacterium tumejaciens (hop through sunflower) was in- 
oculated February 1, 1916, by needle pricks into the very young ovary. Two 
locules are infected, the others are normal. No shoots developed. Orange VI, 
fixed March 10, 1916. Slide 1198A6, stained with acid fuchsin and methyl green. 
The tumor tissue stains red. Actual diameter of the section (short way) }'2 inch. 
Photographed with 75 mm. planar. 



454 



BACTERIAL DISEASES OF PLANTt 




Fig. 345. — Detail of Fig. 344i^ in tumor region. Free locule a,t left; infected 
locule at right; s, young seed the pedicel of which, here torn away, arises from the 
wall at X, a few sections above this one; /, t', tumor tissue filling the locule. Between 
t, t' and X on other sections the tumor tissue is continuous. It appears to have 
developed from the left side of the loculus out of an appendage like h. 



THE CROWN gall: CULTURAL CHARACTERS 



455 




Fig. 346. — Same as Fig. 345 but from the right side of the locule and further 
enlarged. Normal septum at right. The rest is deep-staining tumor-tissue. 
Slide 1198A6; acid fuehsin stain. There are tracheids in the middle of this tumor 
to the left of the part here shown. Observe also the disorderly arrangement of the 
tumor cells. 



456 



BACTERIAL DISEASES OF PLANTS 




Fig. 347. — Crown-gall teratoma on sugar-beet. Plant inoculated by Nellie 
A. Brown, January 26, 1918, using a sub-culture of Bacterium iumcfaciens plated 
from a rose gall. The main axis of the beet is a long way off in the direction of the 
arrow. All here visible is tumor. Growing out of the rough tumor tissue at 
X is a small, bright-green, branched, fleshy (tumefied) shoot. Photographed 
April 25, 1918. X 6. 



THE CROWN gall: CULTURAL CHARACTERS 457 

stages as Avell as later ones); growth in nitrate bouillon; Cohn's 
solution; Uschinsky's solution; milk, litmus milk; behavior in 
peptone water in fermentation tubes with various sugars and 
alcohols. Is there any clouding in the closed end? Try also 
various plant juices in fermentation tubes. What acids are 
produced? Test for formic and acetic. Use flasks of river 
water with 1 per cent peptone, 1 per cent dextrose and a little 
calcium carbonate. Examine at the end of 2 weeks, 6 weeks and 
3 months. According to the chemists, aldehyd, alcohol and ace- 
tone are also produced. Determine its nitrogen nutrition. 

Non-nutritional Environment. — Maximum, minimum and 
optimum temperatures for growth. Thermal death-point in 
jjeptone bouillon (10 minutes exposure). Effect of sunlight, of 
dry air, of freezing, of salted bouillon, of chloroform in bouillon, 
of acids, of alkali, of germicides. Determine conditions under 
which the involution forms are produced. Add various dilute 
organic acids (1 part acid to 9 parts water) in small quantity 
(5, 10, 15 and 20 drops to each 10 cc.) to 24-hour agar and bouil- 
lon cultures (holding check tubes), and compare for changes in 
structure of the organism, i.e., appearance of involution forms 
(Y-bodies) after 24, 48, 72, etc., hours. Pour plates the 3d, 5th 
and 10th days, from both checks and treated tubes, using care- 
fully measured quantities of the fluids. Any reduction in num- 
ber of organisms in the acidified tubes? Any retardation in 
development of colonies on plates poured from such tubes? 

Oxidases (?) and peroxidases are said to be much more abun- 
dant in the galled tissue than in the normal tissue. Can you 
verify these statements? (See Bull. 213, p. 173.) Does this 
fact have any pathological significance? Is the formation by 
the micro-organism of acids and alkalies of pathological signifi- 
cance? (Consult Jour, of Agr. Research, Jan. 29, 1917.) 

How many distinct strains are there of the crown-gall or- 
ganism? I do not call every isolation of an organism a strain, 
but only such as possess distinct morphological, cultural, 
pathogenic or other characteristics. The two strains I am most 
familiar with are the Paris daisy strain and the Hop strain, but 
there are others. Jensen in Denmark isolated a strain from the 
Paris daisy which is unlike our strain in its serological reaction, 



458 



BACTERIAL DISEASES OF PLANTS 





Fig. 34S. 



THE CROWN gall: NON-NUTRITIONAL ENVIRONMENT 459 

that is, the serum of animals inoculated with it will clump its 
cultures but will not clump the cultures of the American daisy 
strain, and vice versa. 

For THE Disease. Signs. — Period of incubation for 
primary tumor (I have observed well-developed small galls on 
the peach 18 days after needle puncture (Fig. 318), on the al- 
mond in 10 days; and beginnings on the daisy in 5 days. 
Under favorable conditions the beginning of galls on sugar- 
beets may also be seen as early as the 4t h or 5th day. 




Fig. 349. — Flagellate rods of Baclcrium tumefaciens (hop strain) stained 
by van Ermengeni's silver nitrate method. Photomicrographed by the writer. 
X 1000. 

Time required for the development of secondary tumors in 
leaves of the daisy? The shortest time I have observed is 10 
days from the time of stem-inoculation and commencement of 
the primary stem-tumor. Ordinarily, it is longer. For produc- 
tion of secondary tumors inoculate into leaf-traces immediately 
under the petiole in rapidly growing Paris daisy shoots. 

Is the tumor or tumor-strand (which is sometimes visible to 
the naked eye) green or greenish? How do you account for 
this? Is it ever brown or brownish? Is it under pressure? 

Fig. 348. — Top of Impatiens balsamina (the common garden balsam) showing 
"hairjr-root" due to inoculation on July 26, 1916, with the hop strain of Bacterium 
tumefaciens. The stem was needle-pricked in the leaf axils. There was much red 
stain in the tumors and in the roots (red flowered variety) although the leaves 
and stems of this plant elsewhere were pale green. There was no red stain in 
tumors on the stems of white flowered balsams inoculated at the same time. 
Photographed August 22, 1916. Nat. size. 



460 BACTERIAL DISEASES OF PLANTS 

Is it really a growth from the primary tumor, in the sense of a 
pushing in between tissues or only a change in more and more 
distant cells owing to the propagation of a chemical stimulus? 
Sometimes it would seem to be the one and sometimes the other. 
If the cells of the tumor do not change position, how do you 
account for the tissue distortions? Can you find the tumor 
strand in fruit trees? Can you cultivate the parasite from the 
tumor-strand? From the secondary tumors? 

What causes the browning of the cut surface of the tumors? 

Describe the appearance of the tumors. What effect, if any, 
do they have on the rest of the plant. Grow- sugar beets or 

CL ''^ 



) 
> 



r 



7 



Fig. 350. — Y-.shaped bodies of Bacterium tumefaciens from a young, pure 
culture treated with acetic acid. Colony 2, resistant daisy, 4 days on agar, then 
exposed 2 days to 10 drops of acetic acid water (1 cc. acid, 9 cc. water). Smeared 
and stained with Carbol fuchsin, March, 1915. 

tobacco (I used Nicotiana sylvestris) and inoculate the center 
of the big rosette of leaves rather early and observe the results. 
Is there ever stimulating action at a distance from the tumor? 
Observe in some of the photographs thickening of the wood on 
the tumor side of the stem. How do you account for it? Ex- 
amine the plant for stunting, curvatures, changes in color of 
leaves, death of parts, etc. Does the location of the gall make 
any difference? 

Histology. — What is the structure of the earliest visible 
tumors (10 or 15 days from date of needle-pricks) as compared 
with structure of the tissue inoculated? How do you account 



THE CROWN gall: HISTOLOGY 



461 




Fig. 35L — Surface and buried colonies of Bacterium tumefaciens (Rose P) 
on +15 beef-peptone agar. Poured January 26. Colonies up January 3L 
Photographed February 1, 1917. X 9 circa. 



462 



BACTERIAL DISEASES OF PLANTS 




Fig. 352. 



THE CROWN gall: HISTOLOGY 463 

for the small size of the cells? (Young actively growing tobacco 
stems or daisy stems may be used for this purpose, making 
shallow pricks.) How many centimeters from the primary 
tumor to the remotest secondary tumor (using daisy) ? On the 
sunflower shown on Fig, 319, sub. 4, it was 7 inches and on 
another it was 8 inches (time 5 to 6 weeks) but, of course, mean- 
while, there was stretching of the stem. Can ,you demonstrate 
the tumor-strand? Can you demonstrate the pseudo-stem 
structure in any of the secondary tumors occurring in leaves of 
the daisy, the primary tumor being on the stem? Is there any 
real difference between the structure of the secondary tumors in 
leaves and those produced on leaves by direct inoculation? 
Peklo states that he obtained root structure (secondary thicken- 
ings) in tumors on flower stalks of the sugar beet by direct in- 
oculation. Is the browned surface of the tumor composed of 
cork? What is the character of the vascularization of the 
tumor? Compare the number and direction of the vascular 
bundles with those of the normal stem and leaf in daisy — in the 
torus of the sunflower. How do you account for the distortions ? 
For the difference in number of vessels in the two tissues? 
Does the tumor contain spiral vessels as well as tracheids? Are 
these abundant or rare? Normal to it or accidental? In cu- 
cumber leaves which contain spiral vessels and no tracheids I 
obtained crown galls containing tracheids and free from spirals. 
Are cambium and phloem normal constituents of the tumor? 
Can you demonstrate sieve tubes in it? Can you produce 
tumors without wounding the cambium, Are they vascularized? 
In such tumors (Fig. 329) how do you account for the tracheids? 
Are sieve tubes also present? Is there any tendency in these 
tumors toward the production of primitive and undifferentiated 
tissues — structures that occur in early stages of growth, or in 



Fig. 352. — Surface and buried colonies of Bacterium tumefaciens (hop strain) 
from Flask P) on +15 beef-peptone agar at end of 4 days at 25°C. Two buried 
colonies coming to the surface. The surface colonies are smooth and translucent 
glistening. Photographed January L3, 1917. X14. In agar-poured plates made 
from old stock cultures the surface colonies of the hop strain are sometimes very 
unlike those shown on this plate, i.e., they may have a contoured surface and 
a sinuate margin with a radiate mottled internal structure, yet are infectious. 



464 



BACTERIAL DISEASES OF PLANTS 




*^^ V •./- 



N 




Fig. 353. — Section of crown gall in Paris daisy showing spindle-shaped tumor- 
cells. Tracheids at the right. Slide cut and stained by Lucia McCuUoch. Photo- 
micrographed by the writer. Medium magnification. 



THE CROWN gall: HISTOLOGY 



465 











:•%*! 
'*«.*•"' 



■■/ 









^ ■♦ ***'*■ 






..I 




Fig. 354. — Crown gall on Paris dai.sy: Photographed from another part of 
same tumor a.s Fig. 353. Non-malignant part of the tumor at the top. The middle 
and lower part is composed of large-nucleate round cells which are in very rapid 
division. The black dots are the deep-staining nuclei. 



30 



466 BACTERIAL DISEASES OF PLANTS 

related plants? See Figs. 353, 354, 415, 416, 417, 418, and 
Bulletin 255, PL LVI (for the wide medullary ray) for what I 
mean. Toward the production of giant cells? What is a 
giant cell? How does the structure of the teratoid crown gall 
differ from that of the non-teratoid gall? On tobacco internodes 
the writer obtained tumors bearing leafy shoots not only from 
the cambium but also from the protoxylem and from the bark. 
How do crown galls differ in structure from fungus galls? 
From insect galls? From nematode galls? How do you ac- 
count for these differences? For structure of the tumor and 
tumor-strand, cut cross-sections, and longitudinal sections 
of leaves and stems (between tumors and through them) from 
fixed material embedded in paraffin. Stain 6 to 24 hours in a 
2 per cent aqueous solution, of methyl green, and after rinsing 
in water gently so as not to wash off the sections, counter- 
stain 5 to 15 minutes in a 2 per cent aqueous solution of acid 
fuchsin (not basic fuchsin). Then pass very rapidly through 
graded alcohols into absolute alcohol, xylol and Canada bal- 
sam. The right amount of staining should be judged under 
the microscope as it is proceeding. Do not overwash the 
sections. 

Such sections may also be stained in various basic aniline 
dyes to demonstrate absence of the bacteria in the vessels and 
intercellular spaces, but the student will hardly be able to dem- 
onstrate the bacteria in the tissues, i.e., inside the cells, by means 
of aniline dyes, unless he should have better success than the 
WTiter and his assistants have had. They may be demon- 
strated by allowing them to diffuse out of the cut tissues in 
bacteria-free water on slides free from bacteria, i.e., clean 
flamed slides, which should then be dried and stained with 
Ziehl's carbol fuchsin, which should also be free from bacteria. 
Both rods and Y's can be demonstrated in this way. They 
should be studied under the 2-mm. oil immersion objective, 
using a No. 8 or No. 12 ocular. 

Certain bodies which at one time I identified as bacteria are 
best demonstrated in the tissues by cutting small slices (2 mm. 
thick) from young and tender galls and throwing them for 24 
hours into 5 or 10 cc. volumes of a 5 per cent aqueous solution 



THE CROWN gall: HISTOLOGY 



467 




Fig. 355. — Advancing margin of crown-gall tumor m the soft white pith of a 
sunflower disk Tumor hard grayish grpen and exceedingly vascular because 
derived from the very vascular torus (seed -receptacle). The white parts at the 
left are surrounded and compressed pith cells. Slide stained with acid fuchsin 
and counterstained with methyl green. Pith white, vessels (tracheids) blue, 
tumor tissue red. Consult also Jour. Cancer Research Vol. I, No. 2, Plate XIII, 
Fig. 52. 



468 BACTERIAL DISEASES OF PLANTS 

of gold chlorid. They are then washed 3 minutes in water and 
placed for another 24 hours (in the dark) in a 0.25 per cent 
aqueous solution of formic acid. After this they are washed in 
water, passed through graded alcohols into xylol and embedded 
in paraffin in the usual way. Is there not at least a strong prob- 
ability that these rod-shaped bodies stained by the gold chlorid 
and supposed to be the bacteria are only normal constituents of 
the plant cell ^mitochondria)? What are mitochondria? 
(Read a paper by the Lewises in Journal of Anatomy, Vol. 17, 
1915, p. 339.) Have you observed any bacteria in the inter- 
cellular spaces of sound galls? Study the fauna and flora of 
old galls. Can you find Tourney's organism? 

Have you observed any excess of chloroplasts in the tumor 
or in the tumor-strand (daisy)? Any bleaching of tumors or 
shoots from tumors ? Any floral pigment in tumors ? Try Pel- 
argoniums, inoculating the tops of growing plants which are 
nearly ready to develop red blossom buds. Try also red bal- 
sams, inoculating before the flower buds develop. Any starch? 
Any excess of sugar or of enzymes? Consult Figs. 353 to 356 
for structure of the hyperplasial tumor tissue. Fig. 353 shows 
spindle-celled tumor tissue and Fig. 354 shows round-celled 
tumor tissue from the same gall. Fig. 355 shows both the 
crushing and invasive effect of a tumor which is excessively 
vascular, because arising from a very vascular organ — ^the torus 
of the sunflower. In Fig. 356 which is from Ricinus the glandu- 
lar epidermis is also involved. When a tumor is deep seated 
should the pushed-up and thickened cortex, the cells of which 
are normally oriented, be reckoned as a part of the tumor? 
If so, why any more than pushed up skin and muscle? 

Variability. — We have found in various isolations from crown- 
gall of the Paris daisy marked differences in virulence (ability 
to produce galls), and from certain natural tumors on the sugar- 
beet (supposed to be crown gall) none of the many typical 
looking colonies on the agar-poured plates were infectious (we 
tried perhaps a hundred). From other similar looking natural 
beet tumors we obtained a very few infectious colonies, but 
these produced only slow-growing small tumors (Bull. 213, 



THE CROWN gall: VARIABILITY 469 

plate 36). Further studies must be made. In this connection 
read what Jensen says in his Danish paper (I.e.). 

Also two extremely virulent isolations, cultivated for several 
years in my laboratory, gradually decreased in virulence and 
finally lost all power to produce galls. Moreover, differences 
have been observed in the vigor of growth and harmfulness of 
galls occurring naturally on various fruit trees. The subject, 
therefore, is not only one of special interest to the pathologist 
but also one of much complexity and considerable discourage- 
ment to the nurseryman and tree inspector. 

Query: May a gall of little harm to one plant infect a soil 
injuriously for another plant? 

Should galled apple trees be planted on land that might 
later receive peaches, raspberries or grapes? Many such 
queries must be left to the future. The subject is one which 
invites careful and long-continued experimentation on the part 
of various experiment stations and boards of inspection. 

Query: Can you produce the disease on olives? On alligator 
pears? On onions or on garlics? On daisy with the hop 
organism? 

Transmission. — Everything we know about crown gall 
points to wounds as the usual, if not the only way of infection. 
Nothing is known respecting insect carriers. In some cases it 
would seem that the ' ' heeling-in " of sound nursery stock in soil 
containing the organism has served to infect the young trees 
(O'Gara). How is the disease spread above ground on the 
limbs of trees? 

Everything points to nurserymen as the world-wide dis- 
tributors of this disease. Many of their soils are so badly in- 
fected that good stock cannot be grown in them. Mr. Waite 
has shown me young apple trees, in numbers, badly galled on 
the graft and almost or quite free in the stock, so that we could 
come to no other conclusion than that the disease was introduced 
into the nursery on the grafts. I have seen badly diseased pear 
stock that was shipped into the country from France, and badly 
diseased peach trees that were shipped into California from the 
eastern United States, and badly diseased gooseberries that were 
shipped from Iowa to the Atlantic Coast, and badly diseased 
roses that were shipped from Ohio to Florida. These are only 



470 



BACTERIAL DISEASES OF PLANTS 




Fig. 356.— Gland-inoculation of Bacterium tumefaciens on Ricinus at end of 
27 days. The needle penetrated more than one layer of cells but the glandular 
epidermis appears to be involved and is dividing. Slide 118S. 



THE CROWN gall: transmlssion 471 

a few out of many instances of such transfers that ha\'e come to 
my attention in recent years. 

LITERATURE 

Read Bulletins 213 and 255, Bureau of Plant Industry, U. S. 
Dept. of Agriculture (to be had from Superintendent of Docu- 
ments, Government Printing Office, Washington, D. C, price 
40 cents and 50 cents respectively). See also " Bacteria in Rela- 
tion to Plant Diseases," Vol. II, Figs. 24, 26, 28, 29, and Plates 
5a, 56, 7, 8, and 10; Phytopathology, Vol. I, pp. 7-11; and Brook- 
lyn Botanic Garden Memoirs I, 1918, p. 448. 

For suggested relations to cancer see also seven summaries 
by the writer: (1) "Le Cancer est-il une maladie du regne 
vegetale?" in Proceedings of the ler Congres International de 
Pathologic Comparee, held in Pai'is in October, 1912 (Tome II); 
(2) "Cancer in Plants" in Proceedings of the 17th International 
Congress of Medicine held in London, August, 1913 (volume de- 
voted to Section III, General Pathology and Pathological 
Anatomy); (3) ''Studies on the Crown Gall of Plants: Its rela- 
tion to Human Cancer" {The Journal of Cancer Research, April, 
1916); (4) ''Further Evidence that Crown Gall of Plants is 
Cancer" {Science, N. S., June 23, 1916); (5) "Mechanism of 
Tumor Growth in Crown Gall" {Jour. Agr. Res., Jan. 29, 1917); 
(6) "Mechanism of Overgrowth in Plants" {Proc. Am. Phil. 
Soc, vol. 56, 1917); (7) "Embryomas in Plants: Produced by 
Bacterial Inoculations" (The Johns Hopkins Hospital Bulletin, 
Sept., 1917). 

Read C. 0. Jensen, " Unders^igelser vedr^rende nogle svulst- 
lignende Dannelser hos Planter." [Investigations concerning 
some tumor-resembling growths in plants.] Kgl. Veterinaer-og 
Landboh^jskolesAarsskrift, Copenhagen, 1918. Serum laboratory 
No. LIV. This paper embodies ten years' study of crown gall 
from the animal (cancer) pathologist's standpoint. 

Read "Crown Gall Injury in the Orchard " by Dean B. Swin- 
gle and H. E. Morris, Bull. 121, Agr. Exp. Sta., Bozeman, 
Mont., Jan., 1918, pp. 124 to 139, with 6 text figures. Their 
experimental work deals with the effect (injurious) of crown gall 
on apple trees and covers a period of 8 years. 



472 BACTERIAL DISEASES OF PLANTS 

Read also Peklo, ''Ueber die Smith'schen Riibentumoren. " 
Zeitschrift fur Zuckerindustrie in Bohmen. Jahrg. XXXIX, 
5 Heft, pp. 204-219, Feb., 1915. Deals with both Bacterium 
tumefaciens and Bacterium heticolum, using cultures sent by the 
writer to Krai in Prague. 

The crown- gall organism was named Bacterium tumefaciens 
by Smith and Townsend in Science, n. s.. Vol. xxv, April 26, 1907, 
pp. 671-673. 



PART IV 
MISCELLANEOUS 

I. NOTES ON SOME ADDITIONAL DISEASES 

The foregoing methods apply to the investigation of all 
bacterial diseases of plants and in case material is not at hand 
for the study of those diseases treated of in Part III, some of the 
following bacterial diseases may be available and in the hands of 
a good teacher will prove equally serviceable. In passing, I 
might say that I have abundant alcoholic material of several of 
the foregoing diseases which I shall be glad to give out in small 
quantity for the preparation of microtome sections for class 
use and that whenever I can do so I shall also be glad to furnish 
teachers of pathology with pure cultures of the various plant 
pathogenic schizomycetes considered in Part III of this book, 
but cannot promise to furnish photographs, stained slides or 
lantern slides, nor any of the organisms mentioned below\ 

1. Mango Leaf-, Stem- and Fruit-spot — Bacillus mangiferae 
Doidge. 

2. Black Spot and Canker of Plum, Peach, etc. — Bad. 
pruni EFS. 

3. Stripe Disease of Broom corn and Sorghum — Bad. 
andropogoni EFS. 

4. Jones, Johnson and Reddy's Bacterial Blight of Barley — • 
Bad. translucens, J., J. and R. 

5. Bacterial Disease of Banana — Bacillus musae J. B. Rorer. 

6. Lilac Blight — Bad. syringae (Van Hall) EFS. 

7. Wakker's Disease of Hyacinths — Bad. hyaci7ithi Wakker. 

8. Cobb's Disease of Sugar Cane — Bad. vascularu7n Cobb. 

9. Rathay's Disease of Orchard Grass — Aplanohader rathayi 
EFS. 

10. O'Gara's Disease of Western Wheat Grass — Apl. agropyri 
O'Gara. 

11. Woods' Disease of Carnations — Bad. woodsii EFS. 

12. Walnut Blight — Bad. juglandis (Pierce) EFS. 

473 



474 BACTERIAL DISEASES OF PLANTS 

13. Coconut Bud Rot — Some form of Bacillus coll, according 
to John R. Johnston. 

14. Larkspur BUght — Bacillus delphinii EFS. 

15. Alfalfa Stem and Leaf Blight — Bad. medicaginis 
(Sackett) EFS. 

16. Stem Blight of Field and Garden Peas — Bad. pisi 
(Sackett) EFS. 

17. Citrus Canker — Bad. cilri (Hasse) Jehle. 

18. Lettuce Blight — Bad. aptatum Nellie A. Brown. 

19. Metcalf's Soft Rot of Sugar Beet — Aplanohacter teutlium 
(Metcalf) EFS. 

20. Tubercle of Sugar Beet — Bad. hdicolum Smith, Brown 
and Townsend. 

21. Leaf Spot of Begonia. 

22. Leaf Spot of Pelargonium — Bad. erodii Lewis. 

23. Aderhold and Ruhland's German Cherry Blight — 
Bacillus spongiosus Aderh. and Ruhl. 

24. Barss' Cherry Blight of Washington and Oregon (which 
is probably the same as No. 23). 

25. Angular Leaf Spot of Cucumber — Bad. lachrymans 
Smith & Bryan. 

26. Spieckermann's ring rot of Potato. Aplanohacter sep- 
edonicum (Spk.) EFS. 

27. Black Chaff of Wheat — Bacterium translucens var. un- 
dulosum Smith, Jones & Reddy. 

28. Halo Blight of Oats — Bad. coronafaciens Charlotte Elliott. 

29. Leaf Spot of Soy Bean — Bad. glycineum F. C. Coerper. 

30. Velvet Bean Leaf ^pot—Bad. stizolobii (Wolf) EFS. 

31. Celery Blight — Bacillus apiovorus Wormald. 

32. Basal glume rot of wheat — Bacterium atrofaciens Lucia 
McCulloch. 

33. Basket willow disease — Bacillus harai Hori & Miyake. 

34. Tobacco Wildfire — Bacterium tobacum Wolf and Foster. 

II. SUGGESTION OF SUBJECTS FOR SPECIAL STUDY 
LARGER PROBLEMS 

1. The natural immunit}^ of plants. 

2. Acquired immunity in plants. Effect of hybridization. 
Search for resistant species and varieties. 



miscellaneous: sl^bjects for special study 475 

3. Carbon and nitrogen nutrition of the soft-rot organisms. 
Species relations. Toleration of acids and alkalies. 

4. Carbon and nitrogen nutrition of the yellow Bacterium 
(Pseudomonas) group. Species relations. 

5. Field studies of soil relations of Bacterium solanacearum, 
especially to lime, phosphates and potash. 

6. Climatic studies of Bacterium solanacearum. Determina- 
tion of its northern and western extension in the United States. 

7. Geographical distribution of Bacterium solanacearum 
in Europe and in South America. Ditto Africa and Asia. 

8. Exact determination of the causes of the bacterial potato 
rots of Australia and New Zealand^ — of France and Italy. 

9. Comparative studies of the white organisms Bacillus 
tracheiphilus, Bacillus amylovorus, Bacterium andropogoni, Bac- 
terium woodsii, and Bacterium mori. 

10. Does Bacterium andropogoni attack maize as well as 
broom corn and sorghum? 

11. Comparison of the above with Bacillus coli, Bacillus 
typhosus, and other related animal pathogenes. 

12. Comparison of the soft-rot bacteria with B. lactis, B. 
coli, etc., in all their varieties. 

13. Comparative study of the green fluorescent pathogenic 
species. 

14. Studies of sub-species of various pathogenes. There are 
a good many. 

15. Critical study of the chemistry of all the tumor-produc- 
ing-species, Bad. tumefaciens in its varieties, Bad. savastanoi, 
Bad. beticolum, etc., determinations to be made from young, 
middle-aged and old flask-cultures in various media. 

16. Determination of all acids produced by plant pathogens". 

17. Hydrogen-ion content of media as related to growth of 
plant pathogenic species. Conversion of Fuller's scale and 
Clark's scale to PH. 

18. Adaptation of the newer culture media used by animal 
pathologists to plant bacteriology. 

19. New differential media for separating closely related 
forms, such as the various soft -rot organisms and the members of 
the yellow Bacterium (Pseudomonas) group. 



476 BACTERIAL DISEASES OF PLANTS 

20. Comparative studies of Aplanobacter species. 

21. Old world distribution of Stewart's disease of maize. 

22. Methods of distribution of parasitic species on seeds, rhi- 
zomes, tubers, corms and bulbs. Responsibility of the seedsman. 

23. Distribution of bacterial parasites on nursery stock. 
Responsibility of the nurserymen and dealer. 

24. Insect and other animal distributors of diseases and their 
control. "Bacillus carriers" among insects. Man's responsibility. 

25. The flora of rotting potato tubers — in the advancing 
margin of the rot and in remoter parts. 

26. The saprophytic flora following each parasitic disease. 

27. Occurrence of black chaff of wheat in Europe, Asia 
and South Africa. 

28. Occurrence of bacterial barley blight in Europe and Asia. 

29. Occurrence of Elliott's oat blights in Europe and Asia. 

30. Symbiotic diseases: Ardisias, Pavettas, etc. 

31. Favoring organisms — bacterial, fungus, protozoan. 

32. Antagonistic soil organisms. 

33. Cause of tobacco mosaic and other mosaic diseases. 

34. Nature of the sugar-cane disease in Brazil. 

35. Nature of the sugar-cane disease in Argentina. 

36. Nature of the East Indian cane-disease known as Sereh. 

37. Nature of the serious Porto Rican stripe-disease of sugar 
cane. This occurs also in Java and Hawaii. One should plant 
sound cane and avoid rattoon crops. Insects spread it. 

38. Nature of the destructive sugar cane disease of Fiji. 
(H. L. Lyons, Hawaiian Planters Record, Vol. Ill, July, 1910, 
p. 200 and F. Muir, Ihid., p. 186). This causes tumors in stems 
and leaves. 

39. Nature of the bark disease of rubber (Petch: Physiol, 
and Diseases of Hevea brasiliensis, London, 1911, PI. IX). 

40. Nature of Janse's disease of Erythrina trees in Java. 

41. Cultural characters of the bacterial organism causing 
tumors on the Aleppo pine and on other European pines. 

42. Cause of peach yellows. Is it a leptome disease? 

43. Cause of peach rosette and of pecan rosette. Are they 
mosaic diseases? Are they denitrification diseases? 

44. Cause and control of bud-rot of the coconut. 

45. Correlation of tobacco leaf spots. How many are there? 



miscellaneous: subjects for specl\l study 477 

46. Bacterial diseases of cultivated orchids. How many? 

47. Bacterial diseases of ferns; 48. of Algae; 49. of fleshy- 
fungi. 50. Do plants harbor animal pathogens? 

51. Is loss of virulence in cultures ever due to the death of 
an invisible symbiont? If not, why do some organisms lose 
virulence quickly and others retain it for many years? 

smaller problems 

I have suggested many such problems in the preceding 
pages and others will occur at once to teachers and students. 

in. PRODUCTION OF TUMORS IN THE ABSENCE OF PARASITES 

On susceptible species of plants, in the absence of parasites, 
overgrowths of suitable tissues can be brought about in at least 
five waj's: (1) by introduction of irritating foreign substances, 
that is by certain poisons, administered in stimulating rather 
than in killing amounts; (2) by slight freezing; (3) by mechanical 
irritation, that is by woundings; (4) by over- watering in a con- 
fined atmosphere; (5) by semi-asphyxiation with vaseline, etc. 

JVIy attention was first drawn to this subject in 1909 as the 
result of observations on the formation of cell-ingrowths (tyloses) 
in the vessels of plants, especially of those attacked by bacteria, 
and, second, by experiments in 1916 with crown-gall products. 

In the vessels of mulberry shoots attacked by Bacterium 
mori, in various plants attacked by Bad. solaiXacearum and in 
old i:)arasitized stems of Cucurbita, Citrullus and Vitis, tyloses 
are ^^ery common but their cause has remained in doubt. They 
occur also in many other plants but I am speaking only of those 
in which I have studied them. To me everything goes to show 
that they are the host-reaction to by-products of invading organ- 
isms which may not be, necessarily, active parasites. I have 
produced them by pure-culture inoculations with Bad. mori 
in young shoots of the mulberry where they never occur naturally, 
(see ''Bacteria in Relation to Plant Diseases," Vol. II, Fig. 30) 
and also with Bad. solanacearum in very young shoots of the 
potato (Fig. 142). Here it is certainly not the bacteria per se 
that act as the stimulus but their soluble products, since the 
tyloses may occur at some distance from the advancing growth 



478 



BACTERIAL DISEASES OF PLANTS 



of the bacteria. This is also suggested by the fact that vessel- 
ingrowths in great numbers may be produced in the absence of 
parasites, by purely chemical means. The most striking ex- 
hibition of tyloses I have ever seen, a veritable pseudo-paren- 
chyma, was obtained in 1914-1915 by Caroline Rumbold in 
the vessels of chestnut wood, by injecting a }ioo gm. solu- 
tion of lithium carbonate (Fig. 357 to 359). Here the effect 
was quite local both in time and place and there were other 
surprising phenomena, viz., the appearance in the bark of 




Fig. 357. — Cross-section of chestnut wood, spring of 1914, showing hirge 
vessels filled with tyloses. Cut in 1915. Below is unaffected autumn wood of 1913. 
From Caroline Rumbold's chestnut bark injections of spring of 1914 using }ioo 
g.m. Li2C03. Photograph by the writer November, 1916. 16 mm., 4 oc. bellows 
at 35. Reduced H. 



numerous well-developed islands of wood, causing it to bulge 
out (Figs. 360, sub. 6 to 362) w^hile in the normal situation in 
1915 much less than the usual amount of wood was produced 
(Fig. 360 at 3). We may suppose the stimulus to have been 
either the alkali, an excess of carbon dioxide liberated from it, or 
both acting together. The same curious phenomenon — enorm- 
ous thickening of the bark (from 1 cm. to 5 or 10 cm.) with form- 
ation in it of numerous islands of wood — occurs in the brown bast 
disease of rubber trees {Hevea brasiliensis) in the Dutch* East 



miscellaneous: tumors in absence of parasites 479 




Fig. 358. — Chestnut wood of the year 1914 in cross-section, showing tyloses 
in the pitted vessels. Bark injected by Carohne Rumbold in the Spring of 1914, 
with 1.^00 g.m- Li.COs, cut and stained by her in 1915. Photomicrograph by 
the writer November, 1916. 8 mm. Zeiss apoc. obj.. No. 4, comp. ocular and 
bellows at .35 on small upright stand. (See Fig. 55.) 



480 



BACTERIAL DISEASES OF PLANTS 




^ri ; % 



^ / 






4 



f^ 






# 




i 




Fig. 359.— Same as Fig. 358, but from a longitudinal section which passes 
through the middle of a big vessel which is full of the proliferated cells. Wood 
at either side, also portion of a medullary ray. Photomicrograph by the writer. 
Same magnification as Fig. 358. Auerbach's stain (C.R.1914, 6). 



miscellaneous: tumors in absence of parasites 481 

Indies, and here the phenomenon is probably due to the alka- 
line by-products of some undiscovered parasite. I also know 
from my crown-gall inoculations that a true cambium devel- 
oped in the bast of tobacco plants may give rise to tumor tissue 
and to shoots (''Embryomas in Plants," 1. c.) and from these 
results it would seem as though wood and bast respectively must 
be developed from cambium in a slightly alkaline medium and In 




Fig. 360. — ChcstnuT hark iiijectiuM ot 1914 In- Caroline Rumbold showing 
i:i cross-section islands of xylem in the phloem: (1) 1913 wood (free from tyloses); 
(2) 1914 wood (full of tyloses); (3) 1915 wood (free from tyloses); (4) cambium; 
1,5) phloem; (6) islands of wood in the phloem; (7) more phloem; (8) cork: 
(9) injected (killed) area, >^oo g-m. LiaCOs being the substance used. Photo- 
graphed by the writer from a section made and stained by Dr. Rumbold. 

a slightly acid medium and that to reverse the ordinary process 
it is only necessary to change the reaction. If this should prove 
true it would help perhaps to explain certain curious phenomena 
of wood and bast distribution observed in the stems of lianas and 
sometimes in other plants (See Jour. Agr. Res., Vol. VI, No. 
4, Plates XX and XXI). By injecting a solution of sodium 
bicarbonate into cabbage stems I obtained hard woody cylinders 

31 



482 



iACTERIAL DISEASES OF PLANTS 




Fig 361 -Detail in cross-section of the inner one-half of an island of wood 
.N-hich developed in chestnut bark following an injection of Hoo g.m. of Uthium 



miscellaneous: tumors in .\bsexce of parasites 483 

in the pith (Fig. 363) but no islands of wood in the bark. The 
experiment, however, should be repeated especially on plants 
with a thicker and firmer bark. In most if not all of my at- 
tempted bark inoculations the fluid ruptured to the surface and 
escaped. 

As in tj'loses, so in crown gall we are forced to conclude that 
it is not the mere presence of the bacteria in the tissues that 
leads to the overgrowth, but rather the stimulus of certain prod- 
ucts of their metabolism. Theoretical considerations led me 
to ask the chemists of the Department of Agriculture to make 
analyses of flask cultures of Bad. tumefaciens and on the basis 
of their findings I experimented with various plants subject 
to crown gall, using dilutions ffluids. vapors) of irritating sub- 
stances said to be present in the cultures. With these I ob- 
tained manj' striking small overgrowths (hyperplasias) and little 
or no evidence of wounding. Such responses were obtained 
with ammonia TFigs. 364, 365), acetic acid (Figs. 366 to 372), 
aldehyd (Fig. 373), and formic acid (Figs. 374 to 377) — all said 
to be crown-gall products. All these tumors are chlorophyll 
free, even when arising in tissues full of leaf-green. 

Many j^ears ago Hermann von Schrenk shov\-ed that in- 
tumescences could be obtained on cauliflower by the use of 
copper salts, and I have seen them on amaryllis sprayed with 
Bordeaux mixture, but. of course, our interest in artificial hyper- 
trophies and hyperplasias centers chiefly around the question 
of their production with substances which are the bj-products 
of parasites. In my first cases, as already stated, I did not detect 
any killing when vapors or large fluid dilutions were used, but 
with AlacCarty's findings in mind (^la^'o Laboratories 0, that 
in ver}' early stages of breast cancers he was able alwaj^s to 
detect a trace of cell-injury preceding the development of the 
malignant cells, I repeated some of my experiments, studying 

1 For list of Dr. ^SlacCarty's suggestive papers on cancer see references at 
end of the second one I have cited under "Literature."' 

carbotiate by Caroline Rumbold. (See her paper in Jour. .Iot. Phil. Soc.) The 
wood occupies the upper one-half of the field and is bedded against the outer face 
of a row of hard bast fibers, the original location of the cambium from which it 
developed. Photographed by the writer from one of Dr. Rumbold's sections. 



484 



BACTERIAL DISEASES OF PLANTS 




Fig. 362. — Outer part of same wood-island in bast as Fig. 361. The dark 
curved line in the middle is the cambium, the dark parallel lines below it are 



miscellaneous: tumors in .\bsence of parasites 485 

earlier (2- to 3-day) stages of the tumor development (hyperplasias 
on cauliflower leaves resulting from acetic acid sprays) and al- 
ways found, judging by differences in staining, indications of 
at least a few killed cells under the stoma through which the 
acid penetrated (Fig. 378). 

In 1918, Harvey, of the U. S. Department of Agriculture, 
showed that cabbage leaves, exposed to — 3°C., freeze at first 



«;xyvv:-.^^'Wii^„ 







> '■Si"Mt&>»rr^<: 



N 





Fig. 363. — Cabbage pith showing a hard wood}" cvHnder which developed 
after injecting a sokition of sodium bicarbonate. Needle track at A'. Under 
X a little of the normal wood cylinder. 

irregularly in small spots and if the freezing be interrupted at 
the right moment, say at the end of 1 2 hour, and the plants re- 
turned to proper conditions, overgrowths (Fig. 379), which 
judging from his sections, may be either hypertrophies or hyper- 
plasias, develop from such chilled or frozen spots (Fig. 380) 
These frozen spots are visible at once because there is extrusion 



xylem medvdlary rays. The white islands in the upper part of the figure are groups 
of hard bast fibers in cross-section. Photographed by the writer from one of Miss 
Rumbold's slides. 



486 



BACTERIAL DISEASES OF PLANTS 




Fk 364-Ricinus stem in longitudinal section showing tumors produced 
by exposure" to the vapors from 0.2 cc. of a 20 per cent solution oi monobasic 
ammonium phosphate held in a small serum tube resting on the next septum below. 
The unopened internode above shows its base also tumefied. 



miscellaneous: tumors in absence of parasites 487 




Fig. 365. — Small tumors uii under-surface of a cauliflower leaf produced by 
vapor of ammonia water. Exposed for 15 mimites in 10 cubic feet of air space to 
0.5 cc. of 0.90 sp. gr. water of ammonia. Photographed after 9 days. X 4. 



488 



BACTERIAL DISEASES OF PLANTS 




%, 




*»» 



W 




Fig 366 — Under-surface of a cauliflower leaf showing small tumult i^rudueed 
by exposure for M hour to vapors of heated Carnoy solution (acetic acid + 
ethyl alcohol) in 10 cubic feet of air space (temperature 20°C.). Exposure 
begun September 21, 1916. Photographed September 28, 1916. X 10. 



miscellaneous: tumors in absence of parasites 489 

of water from the injured cells and this leads to a change in color, 
but this color difference soon disappears and the leaves appear to 
be normal until the growth has advanced to the second or third 
day. As I have recorded elsewhere (''Mechanism of Tumor 
Growth in Crown gall"), the same transient spotting precedes 
the development of tumors on cauliflower leaves exposed to 
dilute vapors of ammonia (Fig. 381) and is undoubtedly to be 
explained in the same way, as due to loss of water from injured 
cells into the surrounding intercellular spaces. 

Earlier in the same year (1918) Wolf showed that intumes- 
cences may be produced on cabbage leaves by means of a sand- 
blast and ascribes intumescences occurring naturally on cabbage 
plants in the field to sand driven by the wind. In this he 
is unquestionably right. They may also be produced on cauli- 
flower leaves in the hothouse by sandpapering the leaves, 
and on bean plants by various woundings. All of Wolf's figures 
are hypertrophies (Fig. 382). 

Some years ago (1892-93) George F. Atkinson experimented 
with the oedema of the tomato, which is an intumescence (Fig. 
383) and reached the conclusion that on susceptible varieties 
(Fig. 384) oedema may be induced by insufficient light and bad 
ventilation coupled with too much water in the soil, and a soil 
temperature too near that of the air, leading to the accumu- 
lation of acids in the plant and to weak cell- walls, easily stretched 
as water is imbibed. He says: ''When there is an abundance 
of water in the plant these acids draw large quantities into 
the cells, causing the cells to swell, resulting many times in 
oedema." . . . "Ordinarily there is no increase in the number 
of cells." He claims to have produced oedema by forcing an 
excess of water into the plants, but his experiments were few, in 
a place where oedema was naturally ver^^ prevalent, and they 
should be repeated. If he made any experiments to determine 
increased acidity they are not mentioned. 

Sorauer, who, following earlier writers, gave the name of 
intumescences to these wart-like growths which occur at times 
on plants of many species, thought they were "caused by an 
excess of water during a period of low assimilation. " In 
another place he speaks of these formations as due to ''an 



490 



BACTERIAL DISEASES OF PLANTS 




Fig. 367.— Under-surface of a cauliflower leaf 4 days after spraying with acetic 
acid in water (Koo.J- Photographed August 5, 1918. X 4. 



miscellaneous: tumors in absence of parasites 491 

abnormal elevation of temperature and excessive water sup- 
ply," combined with weak illumination. With this view 
Atkinson agrees. In the last edition of his book Sorauer 




Fig. 368.^Cross-section, well above the leaf surface, of acetic acid tumor 
on cauliflower leaf 7 days after exposure. Surface covered bj'^ an epidermis. 
Block 1289. Fixed September 28, 1916. The exposure was for ^ hour in 10 
cu. ft. of air space to vapor from 10 cc. of Carnoy's fluid on a warm bath (about 
65°C.). 8 mm. obj., 4 oc, bellows at 50, and enlarged }'i by engraver. Photo- 
micrograph by the writer. 



places intumescences under diseases due to '^excessive moisture 
of the air." 

Other observers regard strong light as favorable and specifi- 
cally on grape leaves (Viala and Pacottet, /. c.) ''excess of light in 



492 



BACTERIAL DISEASES OF PLANTS 







ZAC 



Fig. 369.— Cross-section of acetic acid cauliflower tumor at level of leaf 
surface (stomata of normal leaf surface above and below). >ioo acetic acid 
water sprayed April 10, 1917. FLxed in Carnoj-, April 17. Slide 133652. 
tumor C. 



miscellaneous: tumors in .\bsenxe of parasites 493 




Fig. 370. — Same series as Fig. 369, but tumor D and from the middle level 
of the leaf. In the center compact tumor tissue, surrounded by normal vessels 
and loose mesophyll of leaf. Slide 1336D3. Top row, 2d section from left. 
8 mm. obj., 4 oc, bellows at 45. Photomicrograph by the writer. 



494 



BACTERIAL DISEASES OF PLANTS 












•^f^ 



j|.%*P% 




Fig. 371.— Middle part of Fig. 370 further enlarged. Normal mesophyll 
cells with large intercellular spaces, above, below and at the left. Tumor tissue 
very compact, like normal embryonic tissue. Photomicrograph by the writer. 



miscellaneous: tumors in absence of parasites 495 








Fig. 372.— Longitudinal section of acetic acid tumor on cauliflower leaf. Tissue 
dead at X. Time, 7 days. 8 mm., 4 oc, bellows at 35. 



496 BACTERIAL DISEASES OF PLANTS 

a humid atmosphere." They say, "It is only during periods of 
the most briUiant illumination and directly under the glass of 
the houses, that the intumescences form in quantity. They do 
not occur in the same greenhouse on leaves which are in a diffuse 
light, or in the shade." 

In commenting on this statement Dr. Hermann von Schrenk 
says, "Observations made in the greenhouse of the Missouri 
Botanical Garden during the present season [1904] on grape 
vines which were covered with these intumescences, fully 
bear out the observations made by Viala and Pacottet. The 
intumescences were formed only on the leaves immediately 
under the glass, while all the leaves in the shade Avere free from 
them." 

Judging from my own observations and experiments, made 
on the potato, neither "insufficient light" nor "brilliant illu- 
mination" has anything to do with the formation of intu- 
mescences, at least with those which are hyperplasias. Also 
they may appear in the absence of any excess of moisture and 
when the ventilation is good. 

Not satisfied with the explanation of intumescences above 
given I made experiments of nw own. After some thinking 
as to how best to begin, it appeared to me that I might imitate 
defective greenhouse conditions on a small scale by enclosing 
vegetation in sealed glass tubes. For this purpose I took 
unshriveled, carefully washed, sound potato tubers of several 
varieties, soaked them for 30 minutes in 1:1000 mercuric 
chlorid water to discourage surface organisms, pared away the 
poisoned surface with sterile knives and cut the remainder into 
rectangular blocks. These blocks were then dropped with 
sterile forceps into sterile cotton-plugged test tubes about an 
inch in diameter and containing at the bottom a wad of cotton, 
wet with 1 to 3 cc. of distilled water. The cotton plugs were 
then shoved down a half inch and the top filled with melted 
sealing wax. In these tubes I obtained the results detailed 
below. 

In 1910, the Russian botanist, P. Wisniewski, called attention 
to the production of intumescences on stems by obstruction of 
the lenticels with vaselin. Five years later (1915) the German, 



miscellaneous: tumors in absence of parasites 497 

1 ' ■'^' 'I^^K' Z 




Fig. 373. — (1), (2) and (3). Small tumors on under surface of cauliflower 
leaves produced by formaldehyde (gas), exposure of February 21, 1917. Time, 
6 and 7 days. X 5. 

(4; Cross-section, showing nature of the overgrowth. The dark green palisade 
tissue is only slightly involved. At top, normal thickness of leaf tissue. From 
an unstained free-hand section. 16 mm., 4 oc, bellows at 35. 
32 



498 



BACTERIAL DISEASES OF PLANTS 





Fig. 374.-Under-surface of a cauliflower leat sp ayed ^-^h forn i aad 
water (Moo) on March 24, 1917. Pl-tographed March 3L Nat^^^^^^ 
Shows small tumors and:white (killed) areas. See pi. oo J. Ag. Res. Jan. 29, 



miscellaneous: tumors in absence of parasites 499 




Fig. 375.— Under-surface of a cauliflower leaf in same series as Fig. 374, 
showing tumors due to formic acid (1 pt. to 100 pts. of water). Second spraved 
plant. Time, 7 days. X 4, nearly. 



500 



BACTERIAL DISEASES OF PLANTS 




Y,o 376 — Undei-surface of cauliflower leaf showing overgrowths due to 
formic 'acid water 0/300). Leaf sprayed March 24, 1917. Tumors not so well- 
developed as on plants sprayed with Koo formic acid water, ^.e., not raised so 
high The verv small round tumors are undoubtedly due to entrance of the acid 
in a minimum quantity through a single stoma. Photographed March 31, 1917. 
X 4, nearly. 



miscellaneous: tumors in absence of parasites 501 






n^'ti^ 




Fig. 377. 



502 BACTERIAL DISEASES OF PLANTS 

E. Schilling, published on the same subject, having repeated 
and expanded Wisniewski's experiments with similar results. 
The writer has also experimented on a number of plants, fig, mul- 
berry, olive, begonia, ginkgo, etc. (Figs. 385A, 386) using 
Squibb 's petrolatum. As soon as the lenticels are obstructed, 
gas interchange, i.e., inflow of air and outflow of carbon diox- 
ide and vapor of water, ceases, or at least is greatly re- 
stricted, and following this (in susceptible species, but not in 
ginkgo) the cells under the lenticels at once begin to divide and 
a considerable cushion of cells (hyperplasia) may develop 
(Figs. 3855, and 387 to 389). 

Earlier than this it was known that the lenticels of potato 
tubers frequently proliferate in very moist earth (Sorauer), 
and also that the cut surface of potato tubers may proliferate. 
Fig. 390 shows proliferations that appeared on a pared sterile 
block of raw potato sealed into a test tube some months earlier 
by Mr. Shapovalov, who gave it to me thinking it might be 
crow^n gall. His experiments were for another purpose and these 
growths occurred in one of his check tubes. We could find 
no organisms in the tissues, either with the microscope or by 
means of agar-poured plates. Their internal structure (Fig. 
391) is a twisted vascular hyperplasia not unlike crown gall. 
I tried to duplicate this in an atmosphere of nitrogen but only 
succeeded in asphyxiating the tissues. It would, I believe, 
be easy to get it with sensitive tubers and exactly the right 
reduction of air space or of oxygen. Possibly it is an abortive 
effort on the part of the flesh of the potato to reproduce the 
whole plant. 

Since the preceding paragraph was written I have repeated 
the experiments (spring of 1919) with pared blocks of raw 
potatoes sealed into a moist, confined air-space and have verified 
my prediction, as may be seen from Fig. 392. The blocks, 
which w^ere some of those already referred to as in cotton- 

FiG. 377. — Structure of small and large tumor on cauliflower leaf sprayed 
with 3'ioo formic acid water. From unstained water mounts: (1) Weaker 
stimulus (stomata less open). (2) Edge of a large outgrowth. The whole tumor 
is about four times the length of the part here shown. Both X 130 circa (8 mm., 
4 oc, and bellows at 35). 



miscellaneous: tumors in absence of parasites 503 






\\ 



St 


1 


B 


1 





5t 



Fig. 378. — A. Cauliflower leaf showing slight sub-stomatal injury preceding 
acetic acid tumors. Time, 48 hours. Slide 1416, No. 23. Acid fuchsin stain 
X 130. 

B. Same as A but after 6 days. Here a minimum of acid entered judging 
from small size of tumor. Slide 1420, No. 1. Acid fuchsin stain. 



504 



BACTERIAL DISEASES OF PLANTS 




Fig 379 —Cabbage leaf showing tumors which followed a slight freezing. Leaf 
thawed in air. Time, about 7 days. X 2 circa. {After Harvey.) 



miscellaneous: tumors in absence of parasites 505 



1 







■<.'■ 











Fig. 380.— Varying structure (hypertrophy and hyperplasia) of frost tumors o" 
cabbage leaves. (After Harvey.) 



506 



BACTERIAL DISEASES OF PLANTS 



plugged test tubes sealed in with sealing wax and resting on wet 
cotton, developed glistening ridges or hummocks of rounded 
cells and long hair-like cells (callous tissue) freely, especially in 
the cambium region of the tubers, also occasionally sprouts, 
and over most of their surface in course of a few weeks a well- 
defined cork-layer under which small hyperplasial tumors 
developed, pushing up the cork and frequently cracking it open ; 








Fig. 381. — Cauliflower leaf tliree-fourths hour after exposure to vapor from 
0.5 cc. strong ammonia water (0.90 sp. gr.) in 10 cu. ft. of air space to show fugitive 
mottling. 

sometimes also abortive buds developed from these tumors or in 
their vicinity (Figs. 393, 394). 

The sealed tube experiments were continued for several 
months and many small tumors were obtained (Figs. 395 to 
399). The nutrient substances in these small blocks of potato 
flesh are soon exhausted and the tumors cease to grow, but, if 
one could feed them, it would seem as if growth should continue 
for a considerable period, and that some of the tumors would 
become large. 

When the pared blocks of potato in the sealed tubes de- 



miscellaneous: tumors in absence of parasites 507 






















'.'^) 







Fig. 382. — From Wolf's paper showing structure of intumescences (hypertrophies) 
produced on cabbage leaves by means of a sand blast. 



508 



BACTERIAL DISEASES OF PLANTS 



veloped shoots, the latter regularly produced, under stomata, 
which were always wide open, small intumescences (hyperpla- 
sias) in large numbers both on the stems and on the leaves (Figs. 
400 to 404) . These intumescences were more abundant or rather 
more conspicuous at 28° to 35°C. (Fig. 405) 
than at 23° to 25°C., but they occurred 
also at the latter temperature (Figs. 406), 
although on some parts of the shoots at 
this temperature a microscopic examina- 
tion was necessary to determine their pres- 
ence (Fig. 407). The former were in bright 
light, and the latter in the dark or rather 
in very feeble diffused light, packed away 
in quinine cans. The other conditions 
were the same, viz., a wet base (the block 
stood on very wet cotton or in water), 
saturated air, diminishing oxygen and in- 
creasing carbon dioxide. I am inclined 
to think, in this case, therefore, that the 
intumescences were due to excessive ab- 
sorption of water, coupled with acid 
stimuli liberated by a disturbed transpira- 
tion, due to a saturated or nearly saturated 
atmosphere. Of course, with temperatures 
near the optimum for growth (as would be 
the case in the top of a hothouse in bright 
light) there would be a more conspicuous 
development of such intumescences than at 
lower temperatures in which growth is 
much slower and in the latter it might re- 
quire examination with the microscope to 
demonstrate the beginnings of intumes- 
cences. On the pared sterile blocks of 
potato one of the most striking of these 
tumors, which was narrowly pediceled and 
covered with membrane, developed as a teratoma (Fig. 408). 
In these various examples it will be observed that the cells 
exhibit all grades of development from simple hypertrophy (Figs. 




Fig. 3 83.— Cross- 
section of a Vermont 
tomato leaf showing- 
marked natural oedema. 
Upper surface at left. 
A few of the palisade 
cells unchanged. {After 
Atkinso7i.) 



miscellaneous: tumors in absence of parasites 509 




Fig. 384. — Cross-section of intumescences on tomato produced, it is said, 
by forcing water into the stems: A. Variety No. 18. Normal tissue at the right. 
B. Variety Lorillard. Normal tissue at the right. [After George F. Atkinson.) 



510 BACTERIAL DISEASES OF PLANTS 

380, sub. 1, and 382 to 384) to marked hyperplasia (Figs. 368 to 
372 and 377). They have one element, however, in common and 
in this they differ from all overgrowths due to active parasites, 
that is, corresponding to the fleeting nature of the stimulus, their 
growth is usually of short duration, whereas tumors due to para- 
sites, because supplied with a continuous stimulating exudate 
from the foreign organism, may continue their development 
indefinitely. The physico-chemical stimuli are, however, I 
believe, much the same in all cases where genuine hyperplasias 
occur. To these let us now turn our attention. 

IV. SPECULATIONS ON THE CHEMICAL AND PHYSICAL STIMULI 
UNDERLYING TUMOR-FORMATION 

Classed according to the number and size of their component 
elements, tumors are of three kinds: (1) simple hypertrophies 
(cell enlargements) ; (2) hyperplasias (cell multiplications) ; and 
(3) mixtures of the two, that is hyperplasias containing giant- 
cells. The size and shape of the cells forming the hypertrophy 
or the hyperplasia differ from tumor to tumor even in the same 
tissue, thus in animals we have round-celled, oat-celled and spin- 
dle-celled, large-celled and small-celled connective tissue tumors 
(sarcomas). The nature of tumors varies also, of course, greatly 
according to the nature of the tissue in which they originate, 
since the cells of each organ have a histology and an inheritance 
of their own. For this reason, connective tissue yields one type 
of tumor, glandular tissue another type, vascular tissue a third 
type and so on. 

An enormous amount of data has been accumulated on tumor 
differences, that is on the gross and minute anatomy of tumors, 
especially of human and animal tumors, because this has been 
the easiest method of approach, but it is not the most interesting 
side of the problem. That lies in quite another field, viz., in 
the field of hypothesis and experiment dealing with their 
etiology. 

All overgrowths, without reference to whether they are due 
to parasites or have developed independently of them, appear to 
me to be singularly alike in their chemical and physical origin 
and physiological requirements, their diverse appearances being 



miscellaneous: stimuli lwderlying tumor-formation 511 

attributable to slight variations in the direction or force of the 
stimuli and to the diverse cell-inheritances, each and every tissue 
responding according to its own specific nature. All tumors 
begin, so far as we know, in injured places^ and, fundamentally, 
I believe all may be regarded as excessive and continually modi- 
fied wound-repair reactions. 

In this chapter I shall deal with the secondary causes of 
tumors and shall endeavor to present my ideas as briefly as 
may be, premising that they are based on experiments and 
that where they pass bej'ond experiment into the field of hypo- 
thesis, no one need be led astray, if he keeps my title in mind. 
The best of any iconoclastic writing in science is not so much 
the new facts it has to offer as the changed outlook it gives, 
which new perspective often leads to renewed important experi- 
ments and to general discussion by many workers. I may claim 
to have contributed, at least, this much toward the elucidation 
of the complex and important problems involved in the origin 
of tumors. 

I may state at the outset that my conception of tumor forma- 
tion involves a loss of water and a change of chemical reaction 
in the cells which are to become tumor cells. This change which 
is toward cell-sap concentration and increased acidit}^ must 
occur, I believe, to give the necessary stimulus to tumor forma- 
tion. The stimulus may be long-continued or fleeting and may be 
brought about, as we shall see, in various ways. I will develop 
the subject as I proceed, and only add here at the beginning 
two other hypothetical postulates, first, that hyperplasias appear 
to me to represent a reponse of cells to oxygen-hunger or 
semi-asphyxiation, and, second, that the t3'pe of cell-response 
in the tumor, that is, whether a simple cell-enlargement with 
mitotic or amitotic nuclear multiplication (a giant-cell), or 
a full karyokinetic nuclear division with a corresponding 
hj'perplasia, appears to depend on whether the partial proto- 
plasmic cell-paralysis involves both nucleus and hyaloplasm, 
or is confined to the latter, leaving the nucleus free to divide by 

1 Of 850 breast cancers studied by MacCarty in the Mayo Laboratories every 
one showed evidences of having been preceded by inflammatory injuries (chronic 
mastitis). 



512 



BACTERIAL DISEASES OF PLANTS 







4.%4^ 



^ 



k 



HI 






i^ft 



^ 




Fig. 385.—^. Under-surface of a young green branch of the common orna- 
mental rubber tree {Ficus elastica) showing outgrowths from lenticels 8 days after 
paintmg it with Squibb's petrolatum. On the 5th day the treatment was re- 
peated. Nat. size, nearly. For section at X see B. 

B. Proliferation (hyperplasia) of semi-asphyxiated lenticel of Ficus elastica. 



miscellaneous: stimuli underlying tumor-formation 513 

mitosis and to form cell-walls. The extent of the cell-enlarge- 
ment will then depend on the amount of water imbibed and that 
in turn will depend on the acidity of the cell sap and on the 
corresponding extent to which the physiological control of imbi- 
bition, exerted by the hyaloplasm, is upset. But cell division 
will be rapid if the paralysis involves only the hyaloplasm. 

Let us take the simplest case first, that of hyperplasias 
developed under obturated lenticels (Figs. 385 to 389). Here we 
may suppose that some air still enters and that some vapor of 
water and gas still escapes, but the gas-exchange is demon- 
strably reduced to a very small fraction of what it was, that is, 
^'apor of water and carbon dioxid cannot now escape through 
these openings as before, and air cannot now enter freely. In 
other words, there is a stasis in the tissues under these openings, 
less entrance of air and less movement of aerated water, with 
more or less oxygen-hunger and with increase in cell-acidity 
(due to products of incomplete oxidation) ; also, owing to root- 
absorption, with increase of turgor pressure and, corresponding 
to these changes, a hyperplasia develops. Subjected to these 
conditions, many plants develop small tumors under the lenti- 
cels. The character of the hyperplasia, whether few-celled or 
many-celled, slow-growing or active, will depend on the nature 
of the tissues, and on the extent to which the lenticels are closed 
and the gas-exchange is interfered with. If there is less and 
less gas-exchange, the acid condition and the oxygen-hunger 
will be proportionately increased and the hyperplasia will be 
very small-celled and active. If there is still considerable en- 
trance of air and exit of aerated water and of gas either through 
imperfect closure of the lenticels or directly through the surface 
of the stem, the hyperplasia will be large-celled and slow-grow- 
ing, and this seems to correspond to the facts observed. 

Every growing cell is in constant need of oxygen — must 
have it at once or die. This is absorbed, it is now^ believed, 
through its whole periphery, either directly from the air or in- 
directly out of the aerated fluid which bathes its surface. If the 

Painted with petrolatum March 18, 1918. Photographed from a free-hand, 
unstained section in water, March 26. Upper part of prohferation torn away 
in making the section. 16 mm., 4 oc, bellows at 45. 
33 



514 



BACTERIAL DISEASES OF PLANTS 







Fig. 3S6.— Young vigorous shoot of Morus alba showing lenticel proUferations 
due to closure of the lenticels by Squibb's petrolatum: {1) and (2). Two treated 
portions— 2 was taken a foot above L (Time 13 days; 2d treatment on 11th 
day but not necessary.) Exposure begun [March 5, 1918. Cut and photographed, 
March 18. X 5. (3) Untreated part, above 2. (4) Untreated part, below 1. 



miscellaneous: stimuli underlying tumor-formation 515 

volume of oxygen offered to such a cell (I am not here thinking 
of anaerobes) is reduced by the abstraction either of water or of 
air, it is plain that the only way the cell has of compensating 
for this reduction of an absolutely necessary substance is by 
cell-division, that is by increasing the area of its oxygen-absorb- 
ing surface, or to put it in other words, by increased respiration 
through the development of a hyperplasia.^ If the amount of 
oxygen offered to the tissues is much below the amount required, 
then the hyperplasia will be fine-celled and active, if it is only a 
little below the needs of the tissues, the hyperplasia will be 
coarse-celled and slow-growung. All the evidence we have, 
enzymic and other, points to increased respiration in tumors of 
all kinds, and their feeble vascularization and correspondingly 
slow and uncontrolled circulation leads to just the stasis neces- 
sary to produce more or less oxygen-hunger. I do not mean 
that there is complete absence of oxygen because in ordinary 
plants and animals that would mean prompt asphyxiation and 
death of tissues. Asphyxiation also occurs often in tumors 
but it is an end term that need not concern us here. What I 
mean is just sufficient reduction of the normal supply of oxygen 
to bring about cell-division for compensatory purposes, i.e., to 
afford a larger oxygen absorbing surface. 

Two factors, at least, may be supposed to enter into this 
semi-asphyxiation hyperplasia under obturated lenticels: (1) 
oxygen-hunger, the cells being no longer bathed freely by air or 
by aerated water in movement toward the lenticel ; (2) increased 
acidity of the cell- ap (from incomplete combustion of carbon 
compounds , leading to more or less paralysis of the proto- 
plasmic membrane (the hyaloplasm which governs intake and 
outflow) with correspondingly increased cell-permeability, allow- 
ing water to escape, and water, sugar and other food-stuffs to 
be brought back into the cells in increased quantities from 

^ The reason the bacterial cell accomplishes work out of all proportion to its 
size is just this, that its oxj'gen absorbing surface is enormously greater in propor- 
tion to volume of protoplasm than that of any other known organism. The surface 
of the rods in a cubic centimeter of bacterial slime, such as we frequently obtain 
in a test tube on our solid media and observe in the plant, represents an oxygen 
absorbing area equal to the surface of an ox. Indeed, we might say that the 
smallest bacteria are almost all surface. 



516 



BACTERIAL DISEASES OF PLANTS 




Fj(, 387.-Cross-section of one of the mulberry tumors shown in Fig. 386. 
It is a hyperplasia: A, normal part; B, chlorophyll band; C, lenticel P-Meration. 
Photographed from an unstained section mounted m water. 16 mm. obj., 4 oc. 
and bellows at 45. 



miscellaneous: stimuli underlying tumor-formation 517 




A 







Fig. 388. — A. Hyperplasia in a lenticel on a young olive shoot due to treat- 
ment (March 4, and several times after that) with petrolatum. Response rather 
slow. From an unstained free-hand section. Photographed March 28, 1918. 
8 mm. obj., 4 oc, bellows at 45. B. Cross-section showing a normal lenticel. 
Olive shoot of same age as A. The dark color under the lenticel is due 
to chlorophyll. 



518 



BACTEEIAL DISEASES OF PLANTS 




lb. 



*■ » * 




# ▼ 



x^ 



I -. 



B 



,.-^''gp^;W ^. J . , 



>»*• 



r* 



Fig. 389.— Experimental intumescence on a silver spotted begonia {Begonia 

corallina lucerna) : . . .,, 

A. Cross-section of internode of a young shoot, six days after pamtmg ^Mth 



miscellaneous: stimuli underlying tumor-formation 519 

the siiiToiiiiding tissues to compensate for the substances re- 
moved by growth as the tumors develop, and especially as they 
rupture to the surface and are not protected against irregular 
loss of water. Possibly there may be a third factor involved, 
viz., excessive turgor, due to the pressure of too much absorbed 
water. 

That there is an excessive movement of food-stuffs center- 
ing in all active tumors is shown not only by the rapid growth 
of such tumors, but also by chemical and microscopic analyses, 
by the overgrowth of neighboring tissues not actually involved 
in the tumor itself but affected by it (see Fig. 319, subs. 5 and 6), 
and, finally, by the starvation of remoter normal tissues. 

AYhat proportion the air dissolved in the circulating fluids 
of the plant bears to the direct intake of air through the lenticels 
or the stomata in furnishing oxj-gen to the cells cannot be stated. 
It is conceivable that in many cases the first or indirect source 
would furnish more oxygen to many cells, especially in very 
3^oung organs, the stomata on which are usually closed and the 
intercellular passages n which are undeveloped, w^hile in other 
cases the second or direct source would be most drawn upon. 
Certainly all the water that enters the transpiring plant through 
its root-system (in the aggregate, an enormous amount), as well 
as all the fluid that circulates in animals, is well aerated and 
bathes all the normal living tissues continuously, but there is 
not much active circulation in tumors, and consequently their 
cells will receive less oxygen from this source than normal cells. 

The hyperplasias produced from the flesh of raw potatoes in 
sealed tubes and those developed under stomata on young shoots 
in such sealed tubes, I would explain in the same way. As 
factors in the production of hyperplasias under the stomata, 
I believe we may eliminate both the decreasing external oxygen 
and the increasing external carbon dioxid in the sealed tubes, 

Squibb's petrolatum. The cells of the lenticel have multiplied and pushed up 
the epidermis but have not yet ruptured it. Photographed March 11, 1918, 
from a water mount of an unstained free-hand section. 16 mm. obj., 4 oc, 
bellows at 52. For well developed intumescences see Fig. 394*. 

B. Same as A, but passing through a normal lenticel. From a free-hand 
unstained section in a water mount. The dark color is due to chlorophyll. Photo- 
graphed March 11, 1918. 16 mm. obj., 4 oc, bellows at 52 (small upright stand). 



520 




BACTEEIAL DISEASES OF PLANTS 






Fig 390.— Photographs showing small tumors developing under the cut surface 
(cork-layer) of a raw potato which was sealed into a very limited air space: (.4) 
Front view; {B) upper surface of A; (C) lower surface of A. The cork-layer is 
ruptured in places. Block enclosed in a test tube May 2, 1917, and sealed m with 
sealing wax. At X one of the tumors has developed a bud. Photo, bept. 25, 1917 



miscellaneous: stimuli underlying tumor-formation 521 




Fig 391.— Section of the outgrowth X on cut flesh of Fig. 3904, showing 
distorted vascular structure of the tumor. At left, below, a few normal cells. 
At iett, above, some cells of the newly formed cork layer. 



522 



BACTERIAL DISEASES OF PLANTS 




Fig. 392. — Block of raw potato showing experimental production of .'^mall 
tumors like the accidental ones of Fig. 390. The pared sterile potato flesh, resting 
in wet cottim, was sealed into a test tube February 7, 1919, kept in the dark 
at room temperature, 22° to 25°C., and photographed April 17. X 5. Small 
shoots were torn away at A', which was the bottom of the block bedded in the 
wet cotton. 



miscellaneous: stimuli underlying tumor-formation 523 




Fig. 393.— Top and bottom (right side) of a pared block of sterile raw potato 
sealed into a test tube on wet cotton February 18, 1919, and kept in dull light at 
room temperatures (22° to 25°C.). The photos show top snd side views of the 
two tumors, both of which have ruptured the cork layer that covered them. At 
A,, shoots are pushing. The lower left, block freshly pared to show the thickness 
of the cork-layer. Photographed March 22. X 5. 



524 



BACTERIAL DISEASES OF PLANTS 




Fig 394.— L 2. Blocks of sterile raw potato sealed into test tubes on wet 
cotton February 18, 1919, kept in the dark at temperatures of 23°-25°C., and 



miscellaneous: stimuli underlying tumor-foemation 525 




Fig. 394*. — Well developed intumescences which formed under lenticels on a 
silver spotted begonia that was painted with Sqviibb's petrolatum April 19 and 
22, 1920. Photo. May 20. X 2. 



photographed March 24, X 5: (IC) A ridge of callous tissue from the cambium 
region, also small nodules. (2) At S stunted shoots pushing from a cambium, 
and at either side hyperplasias rupturing through the cork. (3) Small tumor at 
A' bursting through the cork, at F a naked small nodule. (4) Same as 1-3 but 
sealed in February 8, 1919, unpared and photographed March 29. Small hyper- 
plasias rupturing through the original tough cork layer. 



526 



BACTERIAL DISEASES OF PLANTS 




Fjg 395._Pared, sterile block of an Early Rose potato tuber sealed into 
a test tube on wet cotton March 22, 1919, showing development of small tumors. 
Tube kept in the laboratory in dull light at 23° to 25°C. In these sealed tubes 
there was a saturated atmosphere and, owing to continued respiration of the tuber, 
reduction of oxygen and increase of carbon dioxide with probably some anaerobic 
respiration leading to the production of alcohol and acids. Photographed June 
14, 1919. X 5. 



miscellaneous: stimuli underlying tumor-formation 527 




Fig. 396.— From the same series as Fig. 395. A pared, sterile block of Early 
Rose potato showing development of small tumors from the cut surface. Most are 
broad based but X was narrowly pedicelled. Those at the top and the lower 
left are from the region of the cambium; X and Y are from deeper tissues. 
Photographed June 14, 1919. X 5. 



528 



BACTERIAL DISEASES OF PLANTS 




YiQ, 397.— Portion of an Irish Cobbler potato sealed into a test tube on wet 
cotton April 26, 1919. It shows numerous unruptured and ruptured intum- 
escences, the latter bursting through the surface of the tuber (original strong cork 
layer). Flesh more acid than normal potato flesh, i.e., + 35 on Fuller's Scale. 
Photographed May 14, 1919. X 4 circa. 



miscellaneous: stimuli undeelying tumor-formation 529 




Fig 398.— From same series as Fig. 397, showing ruptured and unruptured 
hyperplasias produced in 18 days under the skin of a potato tuber expose 1 to 
dull light on wet cotton in a confined air space at 25°C. Block sealed into the test 
tul^e April 26, 1919. Photographed May 14. X 5. Acidity of whole flesh + 35. 



^'^^ BACTERIAL DISEASES OF PLANTS 




.. /qqq "^^^--^^"f^"^^ ^^«^.«f sterile potato blocks shown in detail in Figs. 397 
and. 398. Above the cotton plugs were plugs of sealing wax. Condensed vapor 
of water is visible on the walls. For details of the two shoots see Fig 403 



miscellaneous: stimuli underlying tumor-formation 531 

because, as we have seen, intumescences often occur in hot- 
houses where there can be no question of diminished oxygen 
(I mean, of course, that external to the plant) or of increased 
external carbon dioxid. The active factors in the production 
of these tumors must then be diminished internal oxygen and 
increased acidity of the tissues (as shown by titrations), both due 
to the saturation of the ambient air which prevents transpiration 
(which normally by continual movement of absorbed water 
continually bathes the tissues in an aerated fluid) and to water- 
logging of the tissues, which prevents the entrance by way of the 
stomata of sufficient external air for the needs of respiration. 
There is certainly saturation of the atmosphere in my sealed 
tubes as there may be in defective greenhouses and at the same 
time there must be consumption of the oxygen by respiration 
and increased absorption of water by the lower parts of the 
plant (roots in hothouse plants and living cells at the base of the 
raw potato blocks in 1113^ sealed tubes). This absorbed water, 
carrying its dissolved air, is passed on to the shoots rather in 
excess tending to internal saturation and turgor pressure, 
but moving more and more slowly owing to the interrupted 
transpiration. The tissues, especially under the stomata to- 
ward which all these various streams of water are moving, will 
then be very full of a sluggish current from which the insufficient 
oxygen is quickly removed, and hyperplasia must then set in 
as a compensatory measure, or otherw'se there will be asphyxia- 
tion of the tissues. As a matter of fact, a little later, suffoca- 
tion did occur in all the roots and shoots developed in my sealed 
tubes (Figs. 402, 404, 406, 408 D, E) but the death of parts, as 
I have said, is a subsequent matter, an end term, which does not 
concern us here. The reason the hyperplasia sets in only under 
the stomata or lenticels is because here are the youngest most 
sensitive cells. 

After the above was written I discovered that intumescences 
could be produced on potato shoots in two other ways: (1) by 
bacterial destruction of the tops while the roots continue to 
function, and (2) by reducing the intake of w^ater. 

On April 10, 1919, I made inoculations on potato plants 
with Bacillus phytophthorus for another purpose (Fig. 211) and 



BACTERIAL DISEASES OF PLANTS 




F:o. 400.-Bn.all hyperplasias developing i" Pot^^^^^^^^^^^ itratTro::! 
air in bright light at hothouse temperatures (28 -35 ^--^^e snoots 
pared sterile potato flesh sealed into a test tube on wet cotton, March -8, 
At XX, callous tissue. Photographed April 10. X 5. 



miscellaneous: stimuli undeelying tumor-formation 533 




Fig. -401.— Block of pared sterile potato flesh exposed to same Ught, temperature 
and atmosphere as Fig. 400 and in all respects like it, except that the lower 
hyperplasias have fused and ruptured encircling the entire base of the shoot 
Photographed April 10. X 5. 



534 



BACTERIAL DISEASES OF PLANTS 




Fig 402 -Shoot of White McCormick potato from a pared sterile block 
cut from a potato tuber and sealed into a test tube on ^vet cotton, February 8 
1919 and held in very dull light at 23° to 25°C. Base and middle covered with 
Ltumescences, ruptur'ed and unruptured. Shoots and roots dying of asphyxia- 
tion. Photographed March 22. X 5. 



miscellaneous: stimuli underlying tumor-formation 




Fig. 403.— Ruptured and unruptured hyperplasias on stunted shoots of 
potato grown m dull light at 25°C., in the sealed tubes shown on Fig. 399 Time 
18 days. The right-hand block also shows two unruptured hyperplasias below 
the shoot and a rupturing one at the left. These are pushing through the very 
resistant cork surface of the tuber. Photographed May 14 1919 x 5 



536 



BACTERIAL DISEASES OF PLANTS 




Fig 4U4-4 Same series as Fig. 403. Roots beginning to die from asphyxia- 
tion. Shoot covered with widely ruptured hyperplasias. Acidity of shoot + 55 



miscellaneous: stimuli underlying tumor-formation 537 

discovered that intumescences in great numbers (Figs. 409, 410) 
appeared on the base of some of the shoots both above ground 
and below, and not only on the older main axis but also on small 
compensatory side shoots which began to develop as soon as the 
tops were destroyed. On a critical examination (9 days after 
inoculation and 5 or 6 days after the tops were killed) these 
intumescences were found on 14 of the 16 inoculated shoots 
(13 pots) and there were none whatever on the 17 control shoots 
(13 pots). The two shoots which did not show anj'' intumes- 
cences were those least injured by the inoculation and which had 




Fig. 405. — Intumescences (hyperplasias) on a stunted tumefied potato shoot 
grown from a pared sterile block of potato flesh in a stagnant saturated atmosphere 
for 10 days in bright light at 28°-3o°C. Tube sealed March 28. Front and back 
view. Photographed April 7, 1919. 

retained a considerable number of actively transpiring leaves. 
One of the 14 plants bearing intumescences was speciall}^ in- 
teresting in that it bore an uninoculated and uninjured side- 
shoot arising from the niain stem at the surface of the earth. 
This shoot, full of green leaves and transpiring freely, bore no 
intumescences whatever at this time, whereas the green base of 
the main axis, killed to within 2 or 3 inches of the ground by the 

on Fuller's scale, that is, excessive. Tissues fuJl of starch and oxydizing enzymes. 
X 5. The acidity of normal potato juice is about + 20. 

B. Same series as Fig. 403, but intumescences more widely ruptured and tip 
of the shoot asphyxiated. Photographed May 14, 1919. X 5. 



538 BACTERIAL DISEASES OF PLANTS 




Fig. 406. — Pared sterile blocks of Early Ohio potato from sealed tubes of 
March 25, 1919. The swollen shoots are covered with hyperplasia! intumescences 



miscellaneous: stimuli underlying tumor-formation 539 



inoculation and thus suddenly deprived of all its freely trans- 
piring foliage, was covered with intumescences, even close to the 
base of the branch. Some days later the branch also developed 
intumescences especially on its base. All the intumescences 
on these plants were hyperplasias and they were in all stages 
of development, from those requiring a microscope for their 
detection to those well-rounded out and those cracked open. 
Also in all the cases 
which I examined the 
stomata over them 
were wide open as if 
in need of air or bur- 
dened with excess of 
water. These plants 
were in 8-inch pots 
and stood on a cen- 
tral bed in a large, 
well-ventilated hot- 
house cool enough to 
be suitable for cauli- 
flowers and they did 
not receive an excess of bright light because they were 15 feet 
from the glass roof and were shaded from the afternoon 
sun by a tall banana house. The possible effect of the 
bacillus, which is an acid producer, was tested by cutting 
off the top of a part of the 17 check shoots whereupon their 
stubs also developed intumescences but more slowly and 
much less conspicuously. In this instance, therefore, neither 
high temperature, nor bright light, nor feeble light, nor a 
saturated atmosphere had anything to do with the production 




Fig. 407. — Hyperplasia under a stoma in tlie middle 
of a potato shoot. From s of Fig. 406 



ruptured and unruptured, and each is beginning to die from asphyxiation, as are 
also the root-tips. Each block stood on wet cotton, and the tubes were kept in the 
dark at 23°-25°C. The parts of the shoots which did not show the hyperplasias 
to the naked eye, as at s, did so under the microscope and they were alwaj'^s 
under stomata (see Fig. 407). Photographed .4pril 14, 1919. X 5. 



540 



BACTERIAL DISEASES OF PLANTS 




Fig. 408.— Tumor which developed on a pared sterile block of raw potato 
(Irish Cobbler) resting on wet cotton in a tube, sealed Feb. 13, 1919: 



miscellaneous: stimuli underlying tumor-formation 541 

of the intumescences, but only a diminished oxygen supply due 
to stasis of the water current and presumably an increased acid- 
ity of the tissues due to this fact, probably in part also to pres- 
ence of acid by-products of the parasite ; and this is illuminating 
as to the origin of other hyperplasias. 

Finally, intumescences occurred freely under stomata on 
swollen stunted leafless shoots (Fig. 411) developing in April from 
dry potato tubers kept on my laboratory table at 25°C., part in 
open deep glass jars exposed to a north light and the remainder, 
of another variety, enclosed in covered paper boxes. Here again, 
neither high temperature, nor bright light, nor feeble light, nor a 
saturated atmosphere, nor in this case excessive water supply had 
anything to do with their production. In these shoots we know 
that there was a defective transpiration apparatus and we may 
assume that there was a sluggish, feebly aerated water-current, 
and consequently that there was more or less oxygen-hunger. 
These swollen shoots were rather hard and were gorged with 
starch (Fig. 412), showing that there was in them, as there is often 
in crown galls, a great excess of sugar beyond that needed for 
growth. This was also made evident by tests with Fehling's 
solution which showed reducing sugars to be scant}^ in the flesh 
of the mother tubers, but to be very abundant in the shoots 



A. Pediceled, white-shining, smootli tumor covered by an epidermis. It 
arises from the cambium. Photo. March 22. X 2, nearly. On May 2 there 
were also several small tumors at t. 

B. The membrane has ruptured widely across the middle of the tumor from 
internal pressure and a tiny shoot has started from its base in the vicinity of the 
pedicel. Photo. March 3L 

C. Further development of the shoot. The tip of the shoot is green, the roots 
are white and the remainder is pink. The tumor is yellowish white. The sealed 
tube has been, all of the time, in the dark at 23°-25°C. The beginnings of in- 
tumescences are visible on the pink and green parts of the shoot. Photo. April 9, 
1919. 

D. E. Front and back view of Fig. C, three weeks later, xlsphyxiation has 
begun. The intumescences are now distinct. Under D there is a freshly cut 
surface; at X, the newly formed cork-layer is visible. Photo. May 2, 1919. X 5. 
The tissues were not tested for acidity, but the juice of similar intumescent shoots 
from other sealed tubes in this series was strongly acid (-(-75 on Fuller's scale) and 
full of starch. The normal acidity of potato tubers is ± 20. 



542 



BACTERIAL DISEASES OF PLANTS 




Fig. 409.— Intumescences (hyperplasias) on the base of a potato shoot full 
of water and in active growth, the whole top of wiiich was suddenly killed by 



miscellaneous: stimuli underlying tumor-formation 543 

(ten times as plentiful as in the tubers). The shoots also 
contained an excess of oxidizing enzymes and a great excess of 
organic acids — 2 to 3 times as much acid as the flesh of the 
mother tubers.^ The shoots had, therefore, every requisite 
for normal growth except a sufficient water-supply. This lack 
of water prevented elongation and the development of the 
respiring and transpiring organs, and the hyperplasias were 
brought on as the result of the increasing acidity and oxygen- 
hunger of the tissues. The stomata over these hyperplasias 
were always wide open (Fig. 413), and here this condition could 
not have been due to excessive water-supply, but must have been 
due rather to oxygen-hunger or to purely physical tensions. 

We may now consider another and quite different type of 
overgrowth, viz., that found on the roots of a great variety of 
plants. I refer to the tumor due to Heterodera radicicola (the 
common gall-producing eel- worm), which tumor is a destructive 
disease on many cultivated plants. Here the larval forms of 
the parasite live, from the beginning, wholly buried in the root- 
tissues. As soon as the worm is hatched it begins to feed on the 
surrounding cells, and immediately there is a tumor response on 
the part of the infested plant. This response, however, is 
quite unlike that under the obturated lenticels. The tumors are 
irregular, soft and large, and composed chiefly of a few cells 
enormously hypertrophied, each cell containing from a half- 
dozen to twenty or more nuclei (sometimes several hundred), 
which either have divided amitotically or have not pushed ordi- 
nary nuclear division as far as to the formation of cell-walls. 
These giant-cells appear in the earliest stages of the tumor, soon 
after the eel- worms have hatched; and always, in thin sections 
of the very young tumors, one may find the young worms sur- 

'The tests were made as follows: weighed quantities (35 grams) of (1) the 
mixed shoots and of (2) the mixed flesh of the tubers were wrapped in surgeon's 
gauze and crushed in the same manner in a mortar, extracted 10 minutes in 100 cc. 
of distilled water at 80°C., filtered, and immediately titrated. 

Bacillus phytophthorus, down to N. Intumescences also at X and Y on the young 
side shoots. Level of soil-surface at L. White McCormick potato inoculated 
in the top of the shoot April 10 and foliage destroyed as early as April 13 or 14 
(see X, Fig. 211). Photographed April 18. X 2. 



544 



BACTERIAL DISEASES OF PLANTS 




Fig 410 -Other side of the stem shown in Fig. 409 showing ruptured and 
unruptured intumescences (hyperplasias) following destruction ot the top by 
Bacillus phytophthorus. Photographed April IS. X 5. This is an exaggeration 
of a common phenomenon. 



miscellaneous: stimuli underlying tumor-formation 545 

lounded by and closely appressed to the big cells (Fig. 414) but I 
have not actually seen their sucking organ inserted into them. 
I have seen these large multinucleate cells in very early stages 
of gall-development where the worms had been present only a 
few days, but always already the head of the young worm is 
in close contact with the cell-membrane where by means of its 
mouth-parts it is able to feed. From this it might be thought 
that the stimulus to growth must reside exclusively in the saliva 
or other excretion from the mouth-parts of the feeding worm, 
and so long as we had no counter observations this explanation, 
which is not yet altogether excluded, appeared to be reasonable 
and sufficient. But there is in Florida on orange-roots, as Cobb 
has shown, a free-living parasitic nematode, closely related to 
Heterodera radicicola, which also sends its mouth-parts into many 
root-cells, but no tumors result, and the explanation I have to 
offer for this marked difference in response is not that the orange 
cannot respond by overgrowths like other plants, since it gener- 
ally responds quickly to crown-gall inoculations but that, the 
greater part of the body of the orange-root nematode being out- 
side of the plant, the anal excretions are voided into the earth 
and do not reach the tissues, whereas in the Heterodera radicicola 
the anal excretions are voided into the tumor and are, I believe, 
the chief cause of its development. The cells nearest to the 
worm receive the greatest volume of stimulus and in these cells 
not only is the protoplasmic membrane paralyzed so that it 
allows a great influx of water and foodstuffs into the cell, but 
the karyokinetic mechanism of the nucleus is also partially 
paralyzed so that the cell cannot divide and the result is not only 
a repeated mitotic division of the nucleus within the cell (I have 
seen 30 nuclei in a cell and Nemec has seen more than 500 in 
cells of Vitis gongylodes) but also at times amitotic division with 
an enormous growth and stretching of the cell-wall until the 
cell often becomes several hundred times its normal size, gorged 
with water and foodstuffs which serve the worms for nutriment. 
Later there may be nuclear fusions, all the nuclei merging into 
one or several mulberry-like conglomerate masses (Nemec). 
These cells are totally unlike any normal cells of the plant and so 
large that, in thin slices of the gall examined superficially, it 



546 



BACTERIAL DISEASES OF PLANTS 




Fig. 4n.-Hyperplasias on tumefied potato shoots g^;™?;^-*^-^;^^^^^^^^^^ 
without soil or water. Diffused north hght, temperature 25 C. Shoots lull to 
1th sugar, acids and oxyd.mg enzymes. The mtumescences were under wxde 
open stomata (See Fig. 413.) 



miscellaneous: stimuli underlying tumor-formation 547 




111 c, I ^'"'''^ '^^*'°'' *^"°"^^ ^^^ hyperplasia! region shoAvn on Fig 

411. 5i,.stomata. Cortex full of starch grains. The pith was also full. Photo- 
micrograph by the writer from a free-hand section mounted in water. 16 mm 
obj., 4 oc, bellows at 35. 



548 



BACTERIAL DISEASES OF PLANTS 




IiG. 413. — Surface view of two intumescences on a potato shoot (Fig. 411) 
showing over each a central wide open stoma. Photoniicrographed by the writer 
at 5:00 p.m. from unstained w^ater mounts. 



miscellaneous: stimuli underlying tumor-formation 549 

seems as if they must be cross-sections of the worm rather than 
of the host-plant. They have, however, been studied critically 
by Nemec and I have been able to confirm many of his findings. 
What the worms excrete into the tissues we do not know, but 
undoubtedly, judging from the excretions of higher animals, 
their waste products must include both acids and alkalies. Pos- 
sibly the giant-cells are due solely to acid mouth secretions. 
There is no reason why the different parts of the tumor might 
not be due to different secretions, e.g., the general hyperplasia 
to the anal excretions and the very peculiar giant-cells to the 
mouth excretions. We shall come again to this subject of acids 
and alkalies in connection, with overgrowths, when, we discuss 
ammonia tumors, acetic acid tumors, crown galls, etc. 

If my hypothesis is correct, farther away from the feeding 
eel-worm larvae the excreted poisons should be more dilute and 
less active, and as the karyokinetic mechanism of the nucleus 
appears to be more resistant to paralysis than the protoplasmic 
membrane of the cell, since in many tumors of plants and animals 
it is not at all interfered with, and by this I mean that in many 
tumors there are no giant-cells, we should expect to find hyper- 
plasia also in remoter parts of the nematode gall and also in its 
earliest stages, and this is just what we do find (Fig. 414). Also 
in club-root, due to Plasmodiophora brassicae, as may be seen 
from Kunkel's photomicrographs, we find similar phenomena. 
Moreover, in a variety of other galls, such as those due to 
various gall flies (Figs. 415 to 424) there is a curious likeness 
to what occurs in the nematode galls, that is, close to the 
feeding organism where the excreted poisons are most concen- 
trated, there is a stretching of the cells to form the so-called 
'' nutritive layer," which layer is rich in sugar, starch, oil and 
albumen, while farther away from the larval chamber, where 
the diffusing excretions would be weaker, there is always a 
fine-celled overgrowth, also stuffed with foods — starch, sugar, 
etc., arranged in such a manner as to be clearly related to the 
stationary larval cavity or cavities, a hyperplasia not developing 
irregularly, as in. tumors due to eel-worms, fungi or bacteria, 
organisms able to move about and thus to change the direction 
and movement of the stimulus, but always quite regular in its 



550 



BACTERIAL DISEASES OF PLANTS 




i^. 




Fig. 414. — A. Early stage ot a lu'inatudc gall on a sugar beet root. In the 
center the young worm is in place and near its head are two giant cells. Farther 
away is the ordinary hyperplasia! tissue of the gall. B. Same as A l)ut from 



MISCELLAXEOUS: STIMULI UXDERLYIXG TUMOR-FORMATIOX 551 

development and relation to the larval cavit}', so much so as to 
have caused great wonder among cecidologists. In these galls, 
the enlarged cells of the inner layer (Fig. 419 Hy) often contain 
more than one nucleus (Fig. 424), i.e., the mechanism of cell- 
division is more or less upset. 

It is worth while here to mention another type of plant tumor 
in which cell-hypertrophy is a marked characteristic but in 
which the nucleus takes even less part than in the preceding. I 
refer to the root-nodules of legumes and, for the sake of a second 
example, again to the club-root of cruciferous plants, the one due 
to a bacterium, the other to a myxomycete. Here the parasite 
is wholly inside the cells, not outside sending into it feeding or- 
gans, as in case of the eel-worm. In both instances the parasit- 
ized cells become enormousl\' distended and filled with the bodies 
of the parasite, the dividing cells being the remoter, non-para- 
sitized cells. The nucleus is killed early in the invasion and all 
the protoplasm of the cell is finally consumed and hence it 
would seem as if the final enlargement of the parasitized cells 
must be due to growth of the cell-wall uncontrolled b}' the proto- 
plasm or to mechanical stretching, and this seems to be confirmed 
by the fact that remote from these large parasitized cells there is 
little evidence of hyperplasia, such as we might expect if soluble 
cell-stimulating poisons were excreted. There is more or less 
cell-multiplication in each of these tumors, often considerable, 
but in their end terms, at least, these two types of overgrowth 
are as far removed as possible from active hyperplasias, and need 
not long detain us, although at one time much study was put 
upon the club-i'oot of turnips and cabbages (due to Plasmodi- 
ophora brassicae) by various persons who thought they saw in it 
certain resemblances to animal cancer. 

More interesting in every wa}- are hyperplasias produced by 
purely chemical means, such as I have illustrated in Figs. 364 to 
377. We may take for discussion two examples, the acetic acid 
tumors and the ammonia tumors. 

When cauliflower plants are exposed for a short time to vapor 



another part of the gall. Cross-section of the young worm lat A^) surrounded by 
multinucleate hypertrophied cells. Farther away is hyperplasial tissue. Medium 
magnification. 



552 



BACTERIAL DISEASES OF PLANTS 



B 




iFg. 415. — A. Polythalamous very leafy gall on sweetbriar (Rosa rubiginosa) 
due to Rhodites rosae L. B. Vertical section passing through some of the larval 
chambers. The felted mass consists of finelj- dissected green leaves of a type 
totally unlike the normal leaves of the plant. 



miscellaneous: stimuli underlying tumor-formation 553 

of ammonia liberated in small quantity in a closed space, the 
protoplasmic membranes of many cells of the leaf are paralyzed 
and water escapes into the intercellular spaces, the evidence of 
this being a fugitive mottling of the leaf, due to the substitution 
of water for air in certain of the intercellular spaces. This 
mottling due to escape of water into the spaces between the cells 
can be produced in various other ways, e.g., by sulphur dioxid, 
as O'Gara has shown, by chloroform, or simply by a minimum 
freezing, as Harvey has shown. Usually within a half hour the 
mottling of the cauliflower leaves due to slight exposure to vapor 
of ammonia (Fig. 381), disappears and the plant again appears 
to be normal, but it is not, since tumors immediately begin to de- 
velop in the least mottled areas and within a few days are plainly 
visible on the surface of the leaf (Fig. 365). Depending ap- 
parently on the amount of the stimulus, or cell-paralysis, these 
tumors are either hypertrophies or hyperplasias, just as they 
are in frozen spots on the same plant and in the insect, nematode, 
and mj^xomycete galls already referred to. This, at least, is the 
way I would interpret it. If the karyokinetic mechanism of the 
cell is paralyzed the hyaloplasm is certain to be more so and 
in the end there will be an inflow of water with stretching of 
the cell (hypertrophy) ; if on the contrary, only the protoplasmic 
membrane is disturbed, with the returning influx of water and 
foods the cell will divide repeatedly and a hyperplasia will result. 
The irregular spotting of the leaves and correspondingly irregular 
development of tumors depends clearly on the var^-ing degree to 
which stomata are open at the time of the exposure, since the 
alkalin vapor enters the leaf through these minute openings. 
In leaves with the stomata wide open there is always a consider- 
able killing effect, similar to that shown in Fig, 374, whereas in 
young leaves with the stomata closed or nearly closed only a 
stimulating minimum of the poison enters and tumors result.^ 

1 To determine quickly the extent to which stomata are open in various areas 
on the upper and under surface of young and old leaves at different times of day, the 
surface may be painted with petrolatvun, the irregular penetration of which corre- 
sponds exactly to the irregular killing effects of excess of ammonia and of acids. 
The writer stumbled on this method independently but apparently it was first 
used by Emmy Stein {Ber. d.d. but. Ges., 30 Bd. 1912, p. 66). 



554 



BACTERIAL DISEASES OF PLANTS 




Fig. 416. — ^Enlarged view of some of the gall chambers of Fig. 4155 together 
with a few of the abnormal leaves. The nearest approach I have seen to such 
leaves is on the calyx but the dissected calyx-leaves are verj- glandular while 
these are not. Possibly there are leaves of this sort on the seedling or on the an- 
cestors of this rose. 



miscellaneous: stimuli underlying tumor-formation ooo 

The further discussion of the cell response may be left until we 
have considered other cases. 

If cauliflower plants are exposed to vapor of alcohol carrying 
acetic acid, or are sprayed lightly Avith acetic acid water (1 
part of acid to 100 or more parts of water) tumors begin 
to form at once in many parts of the leaf, if it is not too old. 
Here again the entrance of the stimulating substance is through 
the stomata and as a result we may assume, for the present at 
least, that always there is a temporary partial paralysis of the 
protoplasmic membrane, that there is initial loss of water, 
that cell-acidity is increased, that the mechanism of respiration 
and transpiration is disturbed, that consequently, cell-division 
is forced, with consumption of sugars, amino acids and proteids, 
and that subsequently there is a compensatory movement of 
water and food-stuffs into the area from surrounding tissues, 
the result being the rapid development of a hyperplasia. This, 
however, grows only for a week or two, just as in case of the 
ammonia tumors, because there has been no repetition of the 
initial small stimulus such as is continually taking place in crown 
gall and in other tumors due to parasites. If it were possible to 
repeat the stimulus at frequent intervals, undoubtedly large 
tumors would result. 

Although I have studied sections of a good many acetic 
acid tumors I have not seen any evidence of hypertrophy except 
in the earliest stages in cells nearest to the stoma and likely to 
have received the greatest volume of the acid. Always the 
remoter growth is a hyperplasia, as in the examples shown (Figs. 
368 to 372). I have followed the sections in series, from the 
level of the leaf surface down to the bottom of tumors, and 
immediately under them in the depths of the leaf, without 
finding any evidence of killed cells in the deeper parts, but 
always, it seems to me, there is at least some slight evidence 
of injury at the surface of the tumor in its middle part close 
under the epidermis (Fig. 378). The initial growth of the tumor 
is not from the epidermis itself but from cells immediately 
below it. Tumors are more apt to begin in the lower parts of 
the leaf than in the upper parts, possibly because the stomata 
on the lower surface are more apt to be open and are also more 



556 



BACTERIAL DISEASES OF PLANTS 




\ 

Fig. 417.— Monothalamous galls due (?) to Andricus iopiarius Ashmead, on a 
Washington, D. C, oak, said to be Quereus prinus, showing tufts of linear leaves 
resembling the leaves of the willow oak (Q. phellos). Probably what is known m the 
older Uterature as Cynips frondosa. These tufts grow from the tissue immediately 
below the gall. 



miscellaneous: stimuli underlying tumor-formation 557 




Fig. 418. — A. Like Fig. -417 but with many of the hnear leaves removed so as 
to show the smooth white gall which seems to occupy the place of a bud. B. 
Vertical section of a similar gall showing a single larva in place. Planar 
enlargements. 



558 BACTERIAL DISEASES OF PLANTS 

numerous. Sometimes after a few days the whole thickness 
of the leaf is involved, at other times the tumor is confined 
chiefly or exclusively to the loose mesophyll, the palisade tissue 
remaining normal or nearly normal. Always there is a striking 
contrast between the surrounding normal, spongy, chlorophyll- 
bearing mesophyll tissue, in which the intercellular spaces occupy 
roughly about one-third to one-fourth of the whole leaf-space, 
and the tumor, in which the cells are free from chloroplasts and 
very compactly arranged, much as in embryonic tissues. Rudi- 
mentary vascular bundles are developed in these tumors but 
their development does not proceed very far because after 
about two weeks the tumors begin to lose water rapidly and 
shrivel. 

Acetic acid, formic acid, ammonia and aldehyd are all 
capable of causing tumor development and are the substances I 
have experimented with most because these are said to be prod- 
ucts of the crown-gall organism and are probably common 
products of a variety of tumor-producing parasites, and for this 
reason are much more interesting than non-vegetable and non- 
animal products, like copper sulphate, mercuric chlorid, etc., 
with many of which small overgrowths may be produced, 
probably in the same way, that is by partial paralysis of the 
hyaloplasm and nucleus of the cell, leading to loss of water, 
concentration of cell-sap, increased acidity, oxygen-hunger, 
nuclear division and return movement of water and food-stuffs, 
the stimulation resulting in a hyperplasia or a hypertrophy 
according to the extent of the paralysis of the protoplasm. 

Let us now turn to higher types of tumors, those which are 
complex in structure and have a self-centered growth. In 
crown gall, as in olive tubercle and sugar-beet tubercle, large 
overgrowths are produced by the bacteria. It will be sufficient 
here to consider the first named tumor, since it is more highly 
developed and more like animal cancer than either of the others. 

What then is the intimate nature of the shock leading to 
tumor formation in crown gall, or in other words, what is its 
secondary etiology? It is, I believe, first of all, following growth 
of the bacteria and the extrusion of their by-products into the 
tissues, an increased acidity in the parasitized cells and prob- 



miscellaneous: stimuli underlying tumor-formation 559 




Fig. 419.— Vertical section through an oak gall like the ones shown in Fig 
118. together with tissue under it: L, larvae. Hv. hvDertronhierl Irvp,-- ui 



hyperplasial layer. Planar enlargement. X 25. 



Hy, hypertrophied layer; HI, 



560 



BACTERIAL DISEASES OF PLANTS 





•m 




Fig 420.— Section of Fig. 419 at X, further enlarged: (1) epidermis; (2) 
subepidermal tissue not much stimulated; (3) vascular tissue; (4) hyperplasia! 
tissue coarser than 5; (5) hyperplasia! tissue full of starch, sugar, etc.; (6; the 



miscellaneous: stimuli underlying tumor-formation 561 

ably also in some of their neighbors, or, to express it in another 
way, it is the liberation of an excess of hydrogen-ions in such 
cells. This leads to a whole train of phenomena whereby a 




Fig. 421. — Section of outer une-lialf of Fig. -119 at T. Vascular tissue in 
center. Outer portion of the stimulated (hyperplasia!) layer at bottom. This 
also contains starch but not as much as the inner portion. 

normal cell (young enough to respond) becomes a tumor-cell, 
and is consequently more or less emancipated from physiological 

hypertrophied laj^er directly in contact with the feeding insect; (7) section of the 
larva. Section cut and stained by Lucia McCuUoch. Photomicrograph by the 
writer. Medium magnification, 8 mm. apochr. obj., 4 oc, bellows at i0}'2- Ihe 
inner layers have taken most of the acid fuchsin stain. 
36 



562 



BACTERIAL DISEASES OF PLANTS 




Fig 422 -Tangential section through the middle wall of the oak gall. The 
cent^il' portion of the figure passes through the mner part of the h>-perplas.al 
layer, the top and bottom through its larger-celled outer part. 



miscellaneous: stimuli underlying tumor-formation 563 

control. Later the tumor as a whole might show increased alka- 
linity, since the organism causing it also produces ammonia, and 
changes in the tumor tending toward decay are always on the in- 
crease. Some of the steps in this process remain to be worked 
out experimentally but we may suppose that they exist and occur 
in about the following order: (1) increased acidity (H-ionization) 
due to bacterial excretions; (2) paralysis of the protoplasmic 
membrane leading to increased cell permeability; (3) loss of water 
from the parasitized cells ; (4) concentration of cell sap ; (5) form- 
ation of cell precipitates; (6) disturbance of transpiration; (7) 
disturbance of respiration; (8) increase of peroxidases and in- 
crease of respiration due to (9) repeated karyokinetic cell- 
division during which food-stufTs are consumed; (10) compensa- 
tory return flow of water and food-stuffs into the tumor favoring 
continued rapid growth, which is also favored in later stages 
by continued superficial loss of water. 

A number of more or less well established facts, some of 
which I have already mentioned, point to these conclusions. 
For instance, we know that in flasks of tap-water containing 
peptone and grape sugar the crown-gall organism produces 
ammonia, aldehyd, acetic acid, and formic acid, and there is every 
reason to suppose that if it can produce these substances in a 
flask it can do so in the plant, and that these are the substances 
which cause the development of the hyperplasia, since I have 
shown that each one of these substances is capable of producing 
small hyperplasias when vaporized or sprayed upon susceptible 
plants. Sugar is abundant in crown galls, often so abundant 
that it cannot all be used in growth and consequently a portion 
of it is re-converted into starch and deposited in the tissues, 
much as glycogen for the same reason, is stored in animal can- 
cers. Sugar is very abundant in acid potato shoots giving rise 
to intumescences (p. 541). It is also abundant in the frost 
tumors (Harvey). Peroxidases are abundant in crown galls 
and also in the frost tumors. There is, undoubtedly, acceler- 
ated respiration in all tumors but who can say whether it is a 
cause or a consequence of the abnormal growth? I have indi- 
cated how I think it occurs, namely as a response to oxygen- 



564 



BACTERIAL DISEASES OF PLANTS 




•^ 





Fig 423.-Tangeiitial section of the oak gall passing in the middle part 
througi; the hypertrophied inner layer. The cells contain large nuclei (often 
several) and take a deep red stain with acid fuchsin. Hyperplasia! (food storage) 
layer at either side. 



MISCELLANEOUS 



: STIMULI UNDERLYING TUMOR-FORMATION 565 




Fig. 424.— .4, B, C. Portions of the inner (hypertrophiedj layer of the oak 
gall highly magnified to show binucleate and multinucleate cells. In A at x 
there is a' nuclear fusion. In C at x the nuclear substance is in seven pieces. 
2 mm. apochromatic oil im. obj., No. 4 oc, bellows at about 40. X 850. (.) 



566 BACTERIAL DISEASES OF PLANTS 

hunger, which may result, as I have shown, from various causes, 
parasitic and non-parasitic. 

Thus, whether brought about by direct abstraction of water, 
as thi'ough wounds; or by dry conditions, as in the potatoes on 
nw table; by semi-asphyxiation, as under obturated lenticels; 
by growth of wet tissues in a minimum of very moist air, as in 
my sealed tubes; by direct addition of ammonia, formaldehyd, 
or acids from without, as through stomata; or finally, by the in- 
troduction into the cells of an alkali and acid-forming tumor 
parasite, the end result is the same, viz., the formation of a 
tumor, the type of which (hypertrophy or hyperplasia) depends, 
I think, on the extent of the cell-stimulus, or cell-paralysis; 
and the extent of the growth on whether the stimulus is self- 
limited or is due to a continually multiplying parasite. 

Recently light has been thrown on some of these problems by 
Dr. R. B. Harvey, of the U. S. Department of Agriculture. In 
experiments on the hardening of plants by cold he has shown that 
when cabbages are exposed to — 3°C. for about 30 minutes their 
leaves freeze in spots and intumescences afterward develop 
from these spots. Such frozen spots extrude water into the inter- 
cellular spaces, as shown by the evanescent spotting, and judging 
from his experiments on egg-white plus phenolphthalein and on 
leaves of Coleus, using their cell-sap as an indicator, freezing 
increases the acidit}^, or hydi'ogen-ion content, of such cells. 
This leads to an increased cell-permeability and to the presence 
in the tissues of an excess of sugar and an excess of peroxidase, 
all of which favor growth. 

In experiments made with red cabbages I could not get the 
same result that Dr. Harvey obtained with Coleus. The frozen 
leaves showed no change in color. Because all of the red pig- 
ment is in the surface cells, the leaves were then ground, mixed 
with a measured volume of distilled water and frozen for three 
hours, but again there was no change in color. All of the check 
tubes but one were kept at room temperature and did not change 
in color. One check tube was exposed for the same length of 
time (three hours) in warm water (60°-62°C.) and this one 
reddened decidedly. On freezing the fluid in this tube changed 
color plainh^ but it became bluer not redder. 



miscellaneous: stimuli uxderlyixg tumor-formatiox 567 

There must be also defective respiration and loss of water 
leading to concentration of cell-sap and increased acidity in 
Wolf's wounded cabbages and my wounded cauliflowers already 
referred to as developing intumescences. 

It is possible also that the tumor-producing action of ammo- 
nia is not very unlike that of the acids I have mentioned. It is 
likely that it would combine at once on entering the living cell 
with some one of the acids always present and act as an acid 
salt. The organic acids most likely to be present in plants 
attacked by crown gall are malic acid and citric acid, and the 
ammonia salts of these acids act in the cell, I believe, like acids. 
It is true they both give an alkaline reaction with Congo red or 
methyl orange but these are indicators which, so to speak, ig- 
nore the acid part, being interested only in the ammonia of the 
salt. If we use phenolphthalein as indicator they both give a 
strongly acid reaction, but here, again, the indicator may be said 
to ignore the other partner in the combination, being interested 
onh' in the organic acid. None of these indicators can, I think, 
be depended upon for the purpose required. Litmus is a much 
better indicator for this purpose because it is a plant product 
and both ammonium malate and ammonium citrate, so far as 
tested, react acid to neutral litmus paper. What is still more to 
the point, they both react acid to the hot water extract of the 
anthocyan from red cabbage, which is the same species of plant 
as the cauliflower on which I have produced the ammonia tumors. 
Up to this time I have not been able to find out what organic 
acids occur in cabbages and cauliflowers, but malic acid is of 
almost universal distribution in plants and citric acid is common, 
so that it is probably one of these two. But even if the ammonia 
acts on the cell directly as an alkali there will be afterward, ac- 
cording to my ideas, an increase in acidity due to loss of water 
through the disturbed hyaloplasm, and in any event there will 
be disturbed respiration. 

In the production then of these weak acids and alkalies 
out of place or in excess in certain tissues we have, it would seem, 
the kej' to the whole tumor situation. Given a multiplying and 
feeble parasite, that is, one able to produce these substances in 
stimulating (membrane paralyzing) amounts and at the same 



568 BACTERIAL DISEASES OF PLANTS 

time destitute of any killing excretions, so that it shall be able 
to live on good terms with the host-cell, and you have my con- 
ception of a tumor-parasite, and in the chain of phenomena set 
up by its presence (H-ionization, disturbed respiration, etc.) 
the origin of all tumors which have the power of self-centered 
continuous growth. 

This physico-chemical hypothesis, which does not always 
require the presence of a parasite in animals any more than in 
plants, since we may suppose that there are in animals slow- 
growing benign tumors quite like the non-parasitic growths 
under stomata and lenticels, serves also to explain the develop- 
ment of fetal fragments in tumors and the formation of teratomas 
in the absence of tumors, thus uniting and correlating all types of 
abnormal growth. I have shown for crown gall that when the 
tumor develops under or in a dormant bud, as for example in 
the axil of a leaf, it always contains embryo-fragments and may 
be full of perishable leafy shoots (Figs. 333, 334, 338, 342). 
I have shown, furthermore, that preformed dormant buds are 
not necessar}^ for the production of these embryomas, but that 
they occur whenever the crown gall organism is inoculated into 
a cambium, that is into a tissue containing totipotent or pluri- 
potent cells (Figs. 335, 336, 339A, 340), that a cambium which 
normally produces only bark-cells may under the crown-gall 
stimulus produce also totipotent cells, and that the nature of 
these embryonic inclusions, i.e., the kind of organs included in 
the tumor, such as roots, leaves, stems, flow^er-buds, depends on 
the location of the tumor or, in other words, on the type of 
mother cells reached and stimulated. 

Recently, I have discovered how to cause dormant totipotent 
cells to develop in the absence of tumor cells. This also I 
believe I have accomplished by increased acidity due to the 
abstraction of water (and along with it oxygen) from very young 
tissues. For this purpose I used buds of the proliferous hothouse 
plant, Begonia phyllomaniaca. On this plant, which is probably 
a hybrid, and which is very sensitively balanced to loss of water, 
it is possible to produce a veritable forest of shoots on leaves and 
on internodes by wounding the roots and also directly in the 
lips of wounds if these wounds are made in very young tissues, 



miscellaneous: stimuli underlying tumor-formation 569 

such as the immature red leaves just emerging from their stipule 
sheathes. See Chapter V. 

What relation, if any, these discoveries, especially those re- 
lating to crown gall, may have to the question of the etiology of 
animal tumors must be left for the oncologists to determine. 
My own views, expressed repeatedl}^ during the last ten years, 
are that they have a profound relation, and Jensen of Copen- 
hagen and Borrel of Paris, as well as several cancer specialists 
in the United States, are of the same opinion. The moment one 
has established that there is a cancer in plants due to an obscure 
wound-parasite it becomes illogical and unthinkable that cancers 
in men and animals are of non-parasitic origin. Why should 
Nature have two such diverse ways of reaching the same end? 
The differences between plants and animals are not sufficiently 
fundamental to lead us to expect it. Both are equally subject 
to parasitic diseases. Both are alike in a hundred ways, as 
Claude Bernard first showed, and as I have elsewhere pointed 
out {Science, I.e.). Moreover, no one has formulated a work- 
able non-parasitic hypothesis. In plants, we see that it is easy 
to produce short-lived tumors by means of a single short expo- 
sure to dilute acids and alkalies, but for the production of a 
continuously growing malignant tumor something more appears 
to me to be necessary, to wit: a feeble commensual parasite of 
a special type, an organism that shall continuously furnish 
acid or alkaline substances to the cells in stimulating small 
quantities, but is not able to destroy the cells. This we have for 
plants in the crown-gall bacterium. Whether the cells thus 
originated may then continue to grow in the absence of the 
parasite, as Jensen believes, is a subject for further consideration. 

That no one has yet isolated a micro-organism from animal 
cancers which will reproduce them when inoculated, does not 
weigh heavily with me for several reasons. I recall the history 
of syphihs, of tuberculosis, of hog-cholera, and of a dozen other 
animal diseases, and I am no longer in awe of the animal path- 
ologist. It may be he is as wrong about cancer as he was about 
syphilis prior to Schaudinn, or tuberculosis prior to Villemin 
and Koch. First, the difficulties in the way of isolation may not 
have been ov^ercome. Parasites are often sensitive to slight 



570 BACTERIAL DISEASES OF PLANTS 

differences in culture media, growing well in one medium and 
refusing to grow in another. See, for example, p. 401 dealing 
with the olive tubercle. Here the organism causing the tumor 
grows in an agar made with one peptone and refuses to grow in 
that made with another peptone, or grows in the presence of the 
latter only with much retardation. See also Fig. 160 (p. 217). 
Many such cases are known to me, as they are to every working 
bacteriologist. Second, the organism may have been isolated 
and neglected for some more common form. How often the 
wrong organism has been selected and studied, and by good 
men too, sometimes for years, in case of other diseases both 
of plants and animals! Nothing is more natural than to select 
for study that which appears to be common and constant on the 
poured plates and yet it may be the wrong thing. Third, cancer, 
at least in the narrower meaning of the word, is a dyscrasia of 
which the tumor is simply the end term and we may not hope 
to reproduce it in animals by the inoculation of an organism until 
the living substratum has become suited to it, that is until we 
have reproduced the beginning and middle terms of the dyscra- 
sia. These may depend on inherited tendencies (Slye) ; or on a 
long-standing vicious physiological disturbance, a malnutrition, 
for example; or on the long-continued action of some feeble 
secondary parasite, a streptococcus, for example. Possibly the 
cancer parasite itself is a streptococcus. Certain streptococci 
have many of the necessary characters of such a parasite, viz., 
low visibility in tissues, feeble parasitism and vitality, sensitive- 
ness to slight changes in culture media so that they will not 
grow, ^ ability to produce acids, ability to destroy blood, ability 
to induce vegetations, etc. My assumption is that the carci- 
noma parasite, if there is one, can act only in an organism which 
has gradually become adapted to it, i.e., only in an abnormal 
body. The normal animal body is amply protected against all 
weak parasites by its leucocytes which might be expected to 
pick up and destroy any injected bacterium of as feeble a 
nature as a cancer parasite must be supposed to be, i.e., one 
incapable of destroying the cells. For this reason plants, 

1 The writer was perhaps the first person to observe (1906) that the strepto- 
coccus of endocarditis {S. viridans) is sensitive to sodium chlorid, and will not grow 
in bouillon or nutrient agar made neutral to phenolphthalein by sodium hydroxid. 



miscellaneous: stimuli underlying tumor-formation 571 

which have no leucocytic apparatus, are easier to work with 
than animals and undoubtedly their tumors will continue to 
yield to careful study many facts which must throw inter- 
esting side-lights on animal tumors, and the time is not far 
removed when they will be studied in many laboratories for this 
purpose. 

Neither does it seem to me a valid objection to the above 
argument that cancers of rats and mice in which no parasite 
has been found are easily inoculable, grow rapidly, and soon 
destroy the host animal. Here the case is quite different from 
that of a naked bacterium. The cancer graft carries into the 
body of the mouse or rat a compact mass of cells, hundreds of 
thousands of them, functioning as a unit. If the graft heals on, 
the cancer cannot fail to continue its malignant growth. If, 
however, it were possible to divide this mass of cells into its 
component elements we should then have a suspension of can- 
cer-cells comparable to a bacterial culture. The cancerous mass 
would then have lost not only its unity but its strength, and 
undoubtedly the body would then react to it very differently, 
that is to say, just as it often does to a few cancer cells that 
have drifted away from the main body of the tumor and are 
surrounded by leucocytes and destroyed in a thrombus, or just as 
it does to an injected bacterial suspension of a feeble para- 
site, a cancer parasite let us assume, each separate foreign 
cell being surrounded and overwhelmed by the leucocytes. 
If we could devise a satisfactory method of overcoming the 
resistance of the normal body in our experimental animals, 
a resistance due to leucocytes and anti-bodies, we should then 
have the pre-cancerous stage, about which we hav^e heard so 
much in recent years, and in reality know so little, and it would 
probably be easy to produce tumors in some of them, even with 
crown-gall bacteria. 

How the oncologist approaches the problem of the etiology of 
tumors will depend very largely upon his training. In looking 
toward the solution of the cancer problem, not much is to be 
hoped from such pathologists as have only a descriptive knowl- 
edge of tumors but much from some of the younger biologists, 
especially those who are well trained in bio-physics and bio- 



572 BACTERIAL DISEASES OF PLANTS 

chemistry, for these are the sister sciences which, when joined 
to physiology and pathology, may be counted upon to solve 
the problem of the cause of malignant human and animal tumors. 
The time is ripe and I believe the solution of the problem is very 
near. The elimination of the disease, however, will be much 
more difficult. 

. LITERATURE 

1886. SoRAUER, Paul. Handbuch der Pflanzenkrank- 
heiten. 2te neubearbeitete Auflage. Erster Theil. Blattauf- 
treibung (Intumescentia) pp. 222-227. Berlin, Paul Parey. 
1886. 

1889. Atkinson, Geo. F. Nematode Root-galls. Science 
Contributions from the Agricultural Experiment Station, Ala- 
bama Polytechnic Institute, Auburn, Ala., December, 1889, 
Vol. I, No. 1, Bulletin No. 9, New Series, pp. 177-226, 6 Plates. 

1893. Atkinson, George F. (Edema of the Tomato. Cor- 
nell Univ. Agric. Exp. Sta., Bulletin 53, May, 1893. 

1893. Atkinson, G. F. (Edema of Apples Trees. New 
York (Cornell) Agricultural Experiment Station, Bulletin 61, 
December, 1893. Published by the Univ., Ithaca, N. Y., pp. 
299-302, Figs. 1 and 2. 

1898. TuBEUF, C. VON (see p. 631). 

1904. ViALA, p. and Pacottet, P. Sur les Vermes des feuilles 
de la Vigne. Comptes Rendus des Seances de VAcademie des 
Sciences. Tome 138, Janvier, 1904, pp. 161-163. 

1905. Von ScHRENK, Hermann. Intumescences Formed as 
a Result of Chemical Stimulation. Missouri Botanical Garden, 
16th Ann. Rpt. St. Louis, Mo., 1905, pp. 125-148. 7 Plates. 

1909. SoRAUER, Paul. Handbuch der Pflanzenkrank- 
heiten. Dritte, vollstandig neubearbeitete Auflage, Erster Band. 
Die nichtparasitaren Krankheiten. Fiinftes Kapitel. Ubermas- 
sige Luftfeuchtigkeit, pp. 435-449. Berlin, Paul Parey, 1909. 

1910. MoLLiARD, Marin. Sur quelques caracteres histolo- 
giques des Cecidies. Produites par I'Heterodera radicicola 
Greff. Revue generale de Botanique, Tome 12, Paris, 1900, pp. 
157-165, 1 PI. 

1910. Nemec, Prof. Dr. B. Das Problem der Befruchtungs- 



MISCELLAXEOUS: STIMULI UXDERLYIXG TUMOR-FORMATIOX 573 

vorgiinge unci andere zytologische Frageii. Chapter \1. 
Vielkernige Riesenzellen in Heterodera-Gallen, pp. 151-173. 
18 text Figs. Berlin, 1910. Gebriider Borntraeger. 

1910. "WisxiEWSKi, P. Uber Induktion von Lenticellen- 
wucherungen bei Ficus. Bulletin International de V Academie 
des Sciences de Cracovie. Serie B: Sciences Xaturelles. Xo. 5B, 
:May, 1910, pp. 359-367. 2 Plates, including 11 Figs., 9 of 
which are photomicrographs. 

1914. Cobb, X. A. Citrus-root Xematode. In Jour. Agr. 
Research. Vol. 2, Xo. 3, pp. 217-230, 13 Figs. 

1915. ^IacCarty, Wm. Carpexter. Facts versus Specu- 
lation in the Professional Conception of Cancer. Texas State 
Journal of Medicine. July, 1915. 

1915. ScHiLLixc;, E. Uber hypertrophische und hyper- 
plastische Gewebeswucherungen an Sprossachsen verursacht 
durch Paraffine. In Jahrb. Wiss. Bot. [Pringsheim]. Bd. 55, 
Heft. 2, pp. 177-258, 43 Figs. 

1916. Smith, Erwix F. Further Evidence that Crown gall 
of Plants is Cancer. Science, X. S., Vol. XLIII, Xo. 1121, pp. 
871-889, June 23, 1916. 

1916. RuMBOLD, Carolixe. Pathological Anatonn- of the 
Injected Trunks of Chestnut Trees. In Proc. Amer. Phil. Sac, 
Vol. 55, Xo. 6, pp. 485-493, PI. 15-18. 

1916. Slye, ]Maud. The Inheritabihty of Spontaneous 
Tumors of Specific Organs and of Specific Types in ]\Iice. Studies 
in the incidence and inheritabilit}^ of spontaneous tumors in 
mice. Fifth Report. . The Journal of Cancer Research. Vol. I. 
Xo. 4, October, 1916, pp. 479-502. 

1916. Slye, ^NIaud. The Inheritability of Spontaneous Tu- 
mors of the Liver in ^Mice. Studies in the incidence and inher- 
itability of spontaneous tumors in mice. Seventh Report. The 
Journal of Cancer Research, Vol. I, Xo. 4, October, 1916, 
pp. 503-522. 

1917. Slye, [NIaud. The Inheritance Behavior of Infections 
Common to ^lice. Studies in the incidence and inheritability 
of spontaneous tumors in mice. Xinth Report. The Journal 
of Cancer Research. Vol. II, Xo. 2, April, 1917. pp. 213-238. 

1917. Smith, Erwix F. ^Mechanism of Tumor Growth in 



574 BACTERIAL DISEASES OF PLANTS 

Crown gall. Journal of Agricultural Research, Vol. VIII, No. 
5, January 29, 1917, pp. 165-186. 

1917. Smith, ErwinF. Embryomas in Plants. (Produced 
by Bacterial Inoculations.) Johns Hopkins Hospital Bulletin, 
Vol. XXVIII, No. 319, September, 1917, pp. 277-294. 115 
Figures on 14 Plates and 1 text Fig. Also a repaged separate. 

1918. Wolf, F. A. Intumescences, with a Note on Mechan- 
ical Injury as a Cause of their Development. Journal of 
Agricultural Research, Vol. XIII, No. 4, April 22, 1918, pp. 
253-259. 

1918. MacCarty, Wm. C. Cancer's Place in General 
Biology. The American Naturalist, Vol. LII, Nos. 620-621, 
August-September, 1918, pp. 395-408. 7 Figs, in text. 

1918. KuNKEL, L. O. Tissue Invasion by Plas7nodiophora 
Brassicae. Jour. Agr. Research, Vol. XIV, No. 12, Sept. 16, 
1918, pp. 543-572. 20 Plates. 

1918. Harvey, R. B. Hardening Process in Plants and 
Developments from Frost Injury. Journal of Agricultural 
Research, Vol. XV, No. 2, October 14, 1918, pp. 83-111. 

(See also Literature references under Crown gall. Part III.) 

V. ON THE production OF TERATOSIS IN THE ABSENCE 
OF TUMORS AND OF PARASITES 

For several years I have been experimenting on the cause 
of excessive proliferation in plants, using for this purpose 
Begonia phyllomaniaca (Fig, 425), and have obtained results 
of general interest which may be expressed as follows, premising 
that the plant is one that has been in cultivation for a long time, 
is of doubtful origin and has been known since it was first 
described by von Martius (in 1852) to throw adventive shoots 
irregularly from its stems and leaves (Figs. 426, 427). It is 
figured and described in Curtis' Botanical Magazine (Vol. 
XVII, pi. 5254), in von Martius' ''Flora Brasiliensis " (Vol. 
IV, Part I, p. 386, Pis. 99, 100), and in various other places. 
Von Martius says the plant was received at the Royal Botanic 
Garden in Munich from a garden in Hamburg about the year 
1848, without name and as of Brazilian origin, but he points 
out that it belongs to a group of begonias which do not occur 



miscellaneous: experimental teratosis 575 

in Brazil. He says it grows frequently in Mexico and Central 
America and thinks De Candolle may be right in supposing 
it to be of Guatemalan origin. 

Bateson believes the plant to be a hybrid because it is 
perfectly sterile, because he has seen phyllomania in sixteen 
hybrids resembling this plant, which he obtained by crossing 
Begonia heracleifolia with Begonia poly ant ha, and because 
Duchartre reported phyllomania in hybrids which Nodot 
obtained by crossing Begonia incarnata and Begonia lucida. 
Bateson thinks the phyllomania cannot be ascribed to the 
sterility of the plant because he knows another begonia called 
Wilhelma '* which is exactly B. phyllomaniaca and equally sterile, 
though it has no trace of phyllomania." He says: ''We 
would give much to know the genetic properties of Begonia phyllo- 
maniaca.'' Goebel thinks the plant was probabh' obtained 
by crossing B. manicata with B. incarnata. His plant showed 
a rhythmic production of the phenomena, the shoots appearing 
only in the winter season, but this, I am inclined to think, 
was only because his gardner repotted the plant in the autumn 
rather than in the spring. 

The main facts I have discovered are briefly as follows : 

1. The initial impetus to phyllomania on a given leaf or 
internode is not determined by the amount of photosynthetic or 
other material in the organ, nor is the phyllomania a low-growth 
or winter state of the plant, as believed by Goebel, but is con- 
ditioned on a definite shock. Whether the phenomenon ap- 
pears in the summer or in the winter depends on when the shock 
is administered. The phyllomania may be produced at any 
time of year. 

2. Following such a shock, however, the number of shoots 
which develop or remain abortive appears to depend on the food- 
supply. 

3. Leaves and internodes are susceptible to shock only 
during a brief period of meristematic growth. When they have 
passed much beyond this period they are no longer susceptible. 

4. The tissues are most susceptible after they have passed 
out of their most primitive condition, but are still embryonic. 



576 



BACTERIAL DISEASES OF PLANTS 




Fig. 425. — A.^^Begonia phyllomaniaca (top removed). Flowers at F. The 
only leaf showing many shoots is X; ^5 of the total leaf surface here shown yielded 
^f of the total number of shoots. Photographed April 13, 1918. About }i 
natural size. B, C. Cluster of flowers, natural size, the larger are pistillate. 
B belongs on the base of C. 



miscellaneous: experimental teratosis 577 




Fig. 426. — Main axis from three plants of Begonia phyllomaniaca. Right and 
left old proliierous shoots, center a young main axis nearly free from proliferations. 
The left stem bore 425 shoots, the right stem 591. The longitudinal white stripes 
are lenticels. Photographed March 27, 1918. X 1>2- 

37 



578 



BACTERIAL DISEASES OF PLANTS 




Fig 



. 427.-Center of blade of a dwarfed leaf, showing proliferations not 



miscellaneous: experimental teratosis 579 

Either way from this stage the degree of susceptibihty usually 
falls rapidly. 

5. Internodes are most susceptible to shock when they are 
about 1 16 to 1^ inch long, and leaves when the folded 2-stipule- 
covered blades are about 3 4 to ^4 inch long, but responses can 
be obtained from considerably larger leaves (blades l^i inches 
long, or more). The closely wrapped terminal bud (Fig. 42SA) 
usually contains three plainly visible young leaves, each sepa- 
rately stipule-wrapped. The outermost is usually about % 
inch long (Fig, 4285, enlarged) including the petiole. The one 
next above it is usually about ^{q inch long, while the third is 
a mere red speck about He inch long. Above these three young 
leaves are several undifferentiated colorless rudiments. 

6. I can now produce the response (teratosis) on a given 
leaf or internode at will. That is to say, in the summer of 
1918 I made experiments, predicted the results, and two months 
later saw my predictions fulfilled to the letter, not once or 
twice, or on a single plant, but in at least 150 places on 34 
different plants. 

7. The response to the shock is in the form of great numbers 
of adventitious embryo plants covering the surface of the leaves 
and other shocked parts (Figs. 429, 430). Very often the num- 
ber of sporadic plants growing out of a shocked leaf-blade has 
been as many as 500 or 600 and occasionally they have ap- 
peared in much greater numbers (2500 to 4000) the leaves above 
and below (Figs. 430, 433, 440, 446) being free or nearly free 
from shoots. The petioles, likewise, respond freely, some- 
times bearing as many as 500 of these tiny plants (Figs. 429a:, 
436^ .T, 438, 4395, 443, 445^). Also on single internodes re- 
mote from leaf axils I have obtained from 200 to 2000 such 
diminutive plants, the internodes above and below the shocked 
ones being free or nearly free (see Figs. 429, 432, 434, 436, 437, 
439^, 444, 445, 449 sub. 3, and Table II). 

8. Most of these crowded shoots perish after some weeks, 
but a considerable number of them live for months, growing 

restricted to the main veins. Contrast with Figs. 441, 442 and 443. There are 
no shoots on the lower surface. Photographed January 14, 1918. X 4. It was 
appearances like this and those in Fig. 426 which led me to make these experiments. 



580 



BACTERIAL DISEASES OF PLANTS 




Fig. 428. — A. Terminal buds of Begonia ■phyUomaniaca. pt, base of upper 
leaf. Each young leaf is separately stipule-wrapped. It was the leaves and inter- 



miscellaneous: experimental teratosis 581 

slo^Yly and developing several small, green leaves, which may be 
of the ordinary shape or may be variously malformed or grown 
together. They do not separate naturally from the parent 
plant to root freely, like bulblets, neither are they to be re- 
garded as branches, except in so far as they subsequently become 
connected with the vascular system of the mother plant, but 
under very exceptionally favorable conditions, as is well known, 
such adventive shoots can be grown into large plants, i.e., 
when separated artificially from the parent plant, rooted with 
great care in sand, and potted in good soil. Unlike Bryophyllum 
no roots develop from these proliferations as long as they 
remain attached to the plant, nor do they root freely in sand. In 
other words, the shoots are of no use to the plant and their 
development cannot be explained teleologicall}'. 

9. The extent of their development on the plant depends 
very largely on the nutrition of the particular organ from which 
they have sprung. If the leaf, for example, remains small they 
are correspondingly dwarfed, many being scarcely more than 
slightly raised green places (pimples) on the surface of the leaf. 
They are better developed on large, well-nourished, actively 
growing leaves and on such leaves a few shoots, especially 
those over midribs where the water supply is most abundant, 
may develop several leaves with blades an inch or more in 
length. I have grown one such laminar shoot (Fig. 428C) 
into a plant 12 inches high, all its nourishment coming through 
the rooted petiole of the mother leaf, the blade of which soon 
perished. It is, however, a crooked stunted plant. 

10. In earliest stages of development these proliferous shoots 
are very superficial. Unlike the normal axillary buds they have 



nodes. in similar buds which specially responded to the root-stimulus, as described 
in the text and as shown on the following plates. Nat. size. 

B. Same as A but with the outermost 2 stipules removed to show the larger 
of the three folded leaves. X 2. 

C. Plant which developed from an adventive shoot on a leaf blade. (1) 
Rooted petiole. (2) Blade of the mother leaf which soon shriveled. Photo- 
graphed July 15, 1918. 

D. Portion of upper main axis of No. 1, first series, enlarged to show the lenticels 
(spindle-shaped white markings) and the glands (numerous white specks) from 
the base of which most of the adventive shoots arise. X 4. 



582 



BACTERIAL DISEASES OF PLANTS 




Fig. 429.-.4. Mam axis of No. 1, first series, showing proliferous internode 
and petiole {X) : 



miscellaneous: experimental teratosis 583 

no connection with the ordinary vascular system of the plant 
(Fig. 451 A leaf, B stem), but are independent growths (parasit- 
ical implants, so to speak) developing either from scattered hairs 
or from various other places in the epidermis, especially glands 
which abundantly dot the surface of the young stems and the 
very young leaves (Figs. 428Z) and 452X, F). In this stage 
they are separated from the vascular cylinder of the stem (Fig. 
4515) and leaf (Figs. 451 A and 453) by a thick layer of colorless, 
coarse-celled hypoderm. These embryo plants, or new organ- 
isms, as they must be considered, develop a vascular system of 
their own, but those that perish (the vast majority) never suc- 
ceed in connecting this with the normal vascular system of the 
parent plant. Those shoots that persist are the ones that have 
formed a junction with the xylem-phloem of the mother plant. 

11. My experiments show that the surface of this plant, at 
least above-ground and in early stages of its growth, has, rather 
uniformly distributed in it, thousands of germinal or totipotent 
cells, most of which ordinarily remain dormant but which can 
be shocked into development, if the shock is applied early 
enough, that is, while the tissues are still very young. These 
shoots are not the development of preformed buds. They are 
not branches, but independent organisms. 

12. Not only are such insignificant organs as the scattered 
petiolar hairs and the base of stem-glands and leaf-glands, as 
already stated, able to grow out into whole plants, but these 
are the parts most likely to give rise to the proliferation. In- 
deed, I suspect that the trichomes and glands are the only parts 
of the epidermis able to develop these shoots but have not made 
enough examinations to be able to pronounce definitely. Judg- 
ing from my experiments, there is germinal tissue at the base 
of every acicular hair and of every botryose gland. Often sev- 
eral shoots arise from a single trichome base or its vicinity, but 
the trichome itself also may give rise to a shoot (Figs. 436C, 



B. Back side of A. 

C. A branch of No. 1, first series, with shocked internode at I. There is a 
narrow strip of cork in the middle of I. 

These proUferating parts were embryonic, stipule-wrapped tissues on July 24. 
Photographed October 8, 1918. X 1%. 



584 



BACTERIAL DISEASES OF PLANTS 




Fig. 430.— From the main axis of No. 1, first series. Very proJiferous, shocked, 
dwarfed leaf {P) and the next two leaves above it (nearly smooth). The order 
of the leaves above P is stated on the cut. Photographed October 8, 1918. Re- 
duced. The actual length of the very proliferous blade was 5 inches. 



miscellaneous: experimental teratosis 585 




Fig. 431.— Smooth leaves, 3 and 4, above the proliferous leaf, P, of Fig. 430. 



586 



BACTERIAL DISEASES OF PLANTS 




Fig. 432.— No. 6, first series, branch arising underground. X, dwarfed 
proliferous leaf; Y, scar of fallen leaf (probably also dwarfed and proliferous); 



i 



miscellaneous: experimental teratosis 587 

440-B, 449, 450). Apparently, on the internodes the shoots are 
not restricted to the vicinity of the glands, but this may be more 
apparent than real, since the glands disappear early. The very 
young normal tissues of this begonia are red, the wound-repair 
tissue also is red. The epidermis of developed organs is green 
but those parts of the leaf giving rise to the acicular hairs are 
red, as if still embryonic. 

13. I was led to study this curious plant, which is smooth 
rather than conspicuously hairy or scaly (Fig. 425), thinking it 
might throw some light on the origin of teratomas in animals 
and hoping that I might be able to discover some law underlying 
its peculiar and apparently lawless behavior. I had one plant 
at first, from which by cuttings I have propagated many. For 
a long time this plant, which was left undisturbed, behaved much 
like any other begonia plant. It refused to throw new adven- 
tive shoots, although it bore some old ones, and I was much dis- 
couraged, but by persistently experimenting with it I have 
succeeded in making it do wonders. The history of my plant, as 
far as I have been able to trace it, is that it was propagated from a 
plant descended from one which was received at the Washington 
Botanic Garden more than 18 years ago. The present super- 
intendent does not know its origin but as Mr. Smith, the former 
superintendent, was a Kew man, I suspect it to have come from 
the Kew Gardens where B. phyllomaniaca has been grown for a 
long time. 

14. In good soil, exposed to medium hothouse temperatures 
and not over-watered,^ the plant grows freely and is healthy. 
There is, however, little substance to it. By this I mean that 
it has a large watery pith, a thick soft cortex and only a thin 
woody cylinder, the amount of water in it being excessive as 
compared with most plants of its size and age. For example, 

^ If watered abundantly the plant in our houses is frequently attacked by a 
fungus (Fusarium sp.) and rots off at the surface of the earth. 

Z, a well developed proliferous leaf; h, c, d, branches of recent origin. The corky 
proliferous internodes are between Y and Z. Their back was free from cork and 
entirely proliferous (Fig. 434, Sub. 2). Z was the small top leaf of July 25, cor- 
responding to leaf A in this photograph. For upper face of Z^ see Fig. 433. 
Photographed October 11, 1918. About 3*4 nat. size. 



588 



BACTERIAL DISEASES OF PLANTS 




Fig. 433.— No. 6, first series. Upper face of leaves Z and Z^ of Fig. 432. Z 
shocked on July 25. Z^ was too old at that time to respond freely. Notice 
regular distribution of shoots in Z and compare with midrib distribution on Figs. 
438, 441, 442, 443. Notice also that the largest leaves are over the main water- 
supply, i.e., over the midribs. About ^<f natural size. 



miscellaneous: experlmextal teratosis 



589 




Fig. 434.— (1) Lower and (2) middle internodes of a shoot of Begonia phyll- 
omaniaca (.\o. 6, 1st series). The middle internodes were induced to proliferate 



590 BACTERIAL DISEASES OF PLANTS 

the above-ground parts of a plant ten months old, 3 feet tall and 
weighing 399 grams, yielded only 28 grams, or 7 per cent, of 
water-free substance, most of w^hich, of course, was the woody 
part. My attention was first called to its watery nature by its 
marked shriveling when put into alcohol, by its prompt disinte- 
gration in boiling water containing acidified copper acetate (I 
could not make a coppered "specimen" of it), and by its exten- 
sive watery hypoderm. It is not known just where begonias 
belong in the natural system, but their general appearance and 
behavior would seem to indicate that they are primitive plants 
or at least hark back easily to primitive conditions. They are 
said to be shade-loving plants, but the only one I have seen wild 
grows on rocks in a tropical sun, and all I have examined under 
the microscope seem to me to show a xerophytic structure. 
Probably some grow in one place and some in another. 

15. I should now state the kind of shocks that cause the 
plant to throw adventive shoots in great numbers. I use the 
expression ''in great numbers" advisedly because the plant is so 
sensitive that it is always throwing more or less adventive shoots, 
especially around the stipule-scars, and I am not speaking of its 
normal behavior but of an abnormal, local and very excessive 
proliferation, as may be seen from my plates. 

I first experimented with stem- and leaf-woundings. These 
are effective, but only if made at the proper time, that is, on 
young tissues. My first experiments made on Y^ to Yi grown 
leaves and internodes failed, probably because such tissues are 
too mature. Later, I got many striking results by wounding 
very immature leaves. My best results were on leaves the 
blades of which were still red and not over an inch long (the 
blades of mature green leaves on w^ell-grown plants are 7 to 10 
inches long) . Here, from the margins of needle-pricks and small 
knife-wounds, where a red callus develops, I obtained dozens and 
scores of adventitious buds, so many, in fact, that often they 
were counted with difficulty (see Fig. 447, where only the larger 

by root injury when these internodes were very small, i.e., wrapped in the bud, 
and the proliferation is ascribed to stimulation due to loss of water. 

3. A branch of No. 1, 1st series, where the proliferation was largelj'' restricted 
by cork-formation which developed in the stimulated internode. 



miscellaneous: experimental teratosis 



591 




Fig. 435.-N0. 8, first series. Main axis. The proliferous internodes are 
15 and 16 and these were embryonic and stipule-wrapped on July 25 An old 
stimulus (due to the May repotting) is shown at 10 and 11, where all but a few of the 
shoots have shriveled and fallen, especially on 11. The leaves p, p, were pro- 
liferous. The leaf N was not proliferous. Most of the shoots were on the left 
side of internodes 15, 16. Photographed October 21,''l918. 



592 



BACTERIAL DISEASES OF PLANTS 




Fig 436.— a. No. 8, first series, third branch, showing proliferous and non- 
proUfeimis internodes. The proliferous internode and leaf above it {x) were 
stipule-wrapped on July 25. Photographed October 14, 1918. 



X IH. 



miscellaneous: experimental teratosis 593 

ones are visible), while the vast mass of the leaf remained free 
or nearly free from such buds. On some leaves, as may be 
seen from Table I, the lips of the wounds bore many times more 
shoots than the rest of the blade, e.g., on the leaf from branch 
I2, }io of the surface (the wounded part) bore 183 shoots while 
the remaining •'^^40 bore only 8 shoots; on the leaf from branch 
nil, If 9 of the surface bore 141 shoots while the remainder bore 
only 5 shoots; on the leaf from branch V2, /is of the surface 
bore 58 shoots, the remainder only 3 shoots. The number of 
shoots from wounds is strictly conditioned on the nutrition. 
Generally each wounded leaf received enough food to enable a 
portion at least of the adventive shoots to develop, but occasion- 
ally not (see Fig. 448Bo and the footnote to Table I). 

16. This local response, however, did not explain the origin 
of extra-axillary buds in the absence of visible wounds and I 
was especially puzzled by the fact that often one or two inter- 
nodes or leaves on a plant would bear hundreds of these adven- 
titious buds (Figs. 426, 427) while all the others wei'e free or 
nearly free from them (Fig. 425, exclusive of x.) 

17. Believing the response must be due to excessive loss of 
water I next tried the drying of cuttings for a short time before 
planting them. In this way, frequently, I obtained more or less 
striking results at the top of the plant, i.e., in the parts which 
were embryonic at the time of making the cuttings, especially 
if the cuttings were allowed to dry for a day or two before plant- 
ing, but this method did not give as uniform results as the follow- 
ing, especially when the drying period was short. 

18. In the spring of 1918 I discovered that the striking re- 
sponse referred to under 16 was due to root-injury sustained at 
the time of repotting. The results of my preliminary observa- 
tions were so convincing that I had no doubt as to the correct- 
ness of my conclusions, i.e., that the very copious restricted or 
regional response was due to root-injury acting on young tissues. 



B. No. 9, first series. Entire main axis (leaves removed). The two proli- 
ferous internodes, at X, Y, were embryonic and stipule-wrapped on July 25. 
For the opposite side of a part of X in more detail see Fig. 439A. Photographed 
October 10, 1918. Reduced. 

C. Shoot arising from a hair on a petiole. The trichome top is shoved over 
to the left and its base is much thickened. X 10. 

38 



594 BACTERIAL DISEASES OF PLANTS 




Fig. 437.— No. 3, second series, main axis. The leaf L, which has fallen, 
V, as the topmost small leaf on July 12 corresponding to Leaf A in the photograph 



miscellaneous: experimental teratosis 595 

In other words, the organs then full grown and proliferous were 
embryonic some weeks earlier at the time of repotting when the 
shock was assumed to have occurred. I reached this conclusion 
by knowing about how much growth a given plant was able to 
make in 6 or 8 weeks and counting back in this way from its tip 
several internodes I would come always to the leaves and inter- 
nodes showing the excessive proliferation of shoots, which leaves 
and internodes must have been quite small at the time of the 
repotting. This, of course, while a legitimate inference, did not 
throw any light on the exact size of the leaf or internode (stage 
of development, wrapped or unwrapped) when the shock occur- 
red and, other than inferentially, did not prove the phyllomania 
to be due to loss of water, nor did it answer the question: Can 
3^ou get it again, or is it seasonal and outside of experimental 
influence? 

19. In July, 1918, therefore, I measured and made records 
of the stage of development of each leaf and shoot on 18 well- 
grown plants before they were repotted, the final records being 
made in the afternoon of July 23 and the plants repotted the 
next forenoon at which time many of the superficial roots were 
cut away. These were plants 10 to 20 inches high (most 13 to 
18 inches) with many fine leaves. They were grown from dried 
cuttings set out March 28, 1918. Every one of them responded 
to the shock more or less, and most of them (all but one dwarf 
plant) strikingly (see Figs. 429, 430, 432, 434, 435, 436, 439.4, 
and first part of Table II). The dwarf referred to was branched 
six times at or near the surface of the earth when examined in 
October and showed no phyllomania on its internodes, but when 
reexamined six weeks later adventive buds in small numbers and 
large corky patches were developing from the proper internodes 
on four of the six shoots. 

20. In a second experiment I used 16 larger plants stand- 
ing on the same bench. These were plants grown from dried 
cuttings planted December 26, 1917. These 16 plants were 



Leaf M (dwarfed) and the internodes Mi and CK were embryonic and stipule- 
wrapped on July 23. Cork has formed at CK. Both M and Mi are full of shoots 
and also CK where it is not covered with cork. L undoubtedly was also pro- 
liferous. Photographed September 30, 1918. Much reduced. 



596 



BACTEEIAL DISEASES OF PLANTS 




Fig. 438. — No. 9, second series, main axis. Upper face of leaf M which was 



miscellaneous: experimental teratosis 597 

repotted July 25, many with root-woundings. Each one re- 
sponded to the shock and most of them (all but two) very 
strikmgly (see Figs. 437, 438, 4395, 440, 443, 444 and second 
part of Table II). It was, in fact, the response of these plants 
to the original drying of the cuttings in December, 1917, and 
to April and May pottings which had given me a clue to the 
cause of the proliferation. Unfortunately for completeness 
sake, it did not occur to me to make Table II until I had ex- 
amined and thrown away a number of the plants without count- 
ing their many proliferations. 

21. In another experiment (Series III, begun after the con- 
clusion of Series I and II because the results were so astonishing 
that I wished additional confirmation) I made use of 29 plants. 
The principal dates are as follows: 

July 12, 1918, cuttings made and left on the bench in dry 
air for three days. 

July 15. Cuttings bedded in sand to root and watered 
sparingly, 

August 6. / Plants set into 4-inch pots. 

September — . Plants set into 6-inch pots. 

November 12. Plants shifted to 8-inch pots where they re- 
mained undisturbed until the close of the experiment. 

March 15, 1919. Experiment closed and plants used for 
other purposes. 

Results: The history of these plants, in which the prolifera- 
tions also occurred in the summer and autumn, not in the winter 
or spring, is as startling as that of Series I and II. During the 
first four months, in which the plants were subject to repeated 
loss of water, from the initial drying and from the root-disturb- 
ances due to three pottings, each one of these 29 plants pro- 
liferated enormously on a great many leaves and internodes 
(Fig. 445). In fact, most of the leaves and internodes, exclusive 
of those which were large when the cuttings were made, prolif- 
erated and many of them bore hundreds of shoots. On the 

an embryonic, stipule-wrapped leaf on July 24. Proliferous petiole at left. Most 
of the 1760 shoots are from the petiole and the vicinity of the midribs. The leaves 
above and below this leaf were comparatively free from shoots. Photographed 
September 27, 1918. About ^^ nat. size. 



598 



BACTERIAL DISEASES OF PLANTS 




Fig. 439.— a. No. 9, first series. Middle part of lower proliferous internode 
(back of X in Fig. 436B). In the center at C a small corky area. Photographed 
October 10, 1918. X 4. 



miscellaneous: experimental teratosis 599 

first of December it did not seem as if the plants would ever 
produce smooth leaves, but during the second four months, in 
which the plants remained undisturbed and were watered spar- 
ingly but regularly and sufficiently, they prohferated either not 
at all or scarcely at all, producing several hundred smooth leaves 
and internodes, so that on March 15 each one of the 29 plants 
closely resembled the plant shown on Fig. 425. 

When finally examined on March 15 nearly all of the lower 
very proliferous leaves^' had fallen (see Table III) but there 
were still a large number of proliferations visible on the lower 
internodes, that is adventitious growths on the lower 5 to 9 
inches of the stem, corresponding to the stimuli of July 12-15, 
August 6, September — , and November 12, but all of the upper 
15 or 20 inches of the plant was so nearly free from adventive 
shoots that one would say entirely free until he examined 
closely, when a few shoots were found here and there on the 
leaves land internodes, but in no case dozens or hundreds, as 
during the first period. Great numbers of the leaves were 
particularly fine and smooth. In other words, undisturbed, 
the plants ceased to proliferate, except around the stipule 
scars which are places more subject, it would seem, to loss of 
water than other parts and which always proliferate except 
on the youngest nodes, upper 4 to 6 inches of the stem (Fig. 
445 St) where the stipules are still living. The above state- 
ments hardly express the full difference, because at the close 
of the experiment (March 15) there were more than 800 smooth 
leaves, whereas on the first of December there was not a single 
one. 

22. It now remains to consider the nature of this shock, 
since merely to say that wounding causes it, or that it is the 
plant's response to a wound, does not satisfy, and especially 
not, because the place of response, as we have seen, may be very 
far away from the place of wounding, i.e., as far as from the 
roots to the top of the plant. I have only a hypothesis to 
offer, but it is confirmed by the experiments, fits in very well 

B. No. 9, second series, main axis. A small portion of the proliferous petiole 
of Leaf M (Fig. 438) enlarged. Nearly ever}' trichome has developed a shoot. 
X 4. 



600 BACTERIAL DISEASES OF PLANTS 

with some other facts, aheady detailed in the preceding chapters, 
and may help to throw light on the mechanism of the response, 
and also, perhaps, on the origin of animal teratomas and of 
certain cancerous phenomena. 

23. First, let us try to conceive what takes place in the 
plant when it is suddenly deprived of a part of its roots by 
cutting or breaking, while others cease to function, owing to 
loosening of the previously compact earth. One thing at least 
happens with certainty, there is a sudden marked diminution of 
the very considerable water-supply necessary for the well being 
of the plant. The plant is transpiring more or less freely over 
a broad leaf surface (several square feet) and suddenly it is de- 
prived of its water-supply. This, I believe, is the shock which 
starts the dormant totipotent cells into development. It is 
impossible that the plant should wholly cease to transpire. 
Since it must breathe to live, water will escape from its stomata 
and lenticels, and it seems likely that the thin-walled, immature 
and delicate tissues of the terminal buds will be just the ones 
from which proportionately most water will be abstracted to 
meet this unusual demand and which will be most shocked by 
the loss they sustain. That the plants lose water in ex- 
cess is demonstrable by the balances. Other phenomena also 
occur but these are secondary results, e.g., there is sometimes 
slight wilting especially in cuttings, growth above-ground 
ceases for a short time and the elaborated food that would 
have been used for further extension of normal shoots is sent 
downward to make new roots. The aeration of the interior must 
also be less perfect than it was since the absorbed water always 
carries dissolved air. Less oxygen, therefore, will reach the 
tissues, and, theoretically, they should become more acid than 
normal.^ 

1 Subsequently I tested the terminal liuds of 42 plants of Series VI with the 
following results: 

A. I. 21 tops (liearing 4-5 leaves) cut from the plants and dried 48 hours at 
28°-30°C. on a laboratory table (July 12-14, 1919) in diffuse light: 

1. Fresh weight, 740.25 grams. 

2. Loss of weight in 48 hours, 191.50 grams. 

3. Juice of the 21 buds crushed at end of the 48 hours, extracted 10 minutes in 
hot water (90°C., falling to 35°C.), squeezed dry in a hand screw-press and filtered: 



miscellaneous: experimental teratosis 601 

There can be no reasonable doubt, therefore, that the tissues 
which afterward prohferate not only lose water, hut become more 
acid. 

In a week or two all the root-injury has been repaired and 
the plant takes on a new and vigorous growth and apparently 
no harm has resulted. This new growth is, of course, from the 
terminal buds which were shocked and the leaves and internodes 
of which will exhibit copious proliferation when they develop. 
As growth continues and the shoot elongates we soon come to 
leaves and internodes formed after the date of the shock and 
these will be free or nearly free from adventive shoots, like the 
lower older tissues which were mature or semi-mature at the 
time of the root-injury. Thus we may have on a large plant 

Acidit.y + 15S (Fuller's scale). The readings of the 3 titrations were L59, 157 
and 158. 

II. 21 fresh terminal buds brought in, crushed, extracted, squeezed and filtered 
as above: 

Acidity + 126 (three titrations, each of which gave the same reading). 

This experiment was repeated some da.ys later (July 22-23) with the following 
results : 

B. I. 9 tops (bearing 4 and 5 leaves each) cut away and left on a table for 24 
hours : 

1. Fresh weight, 288 grams. 

2. Loss of weight in 24 hours, 44.5 grams. 

3. Juice of the 9 buds including uppermost small undeveloped red leaf extracted 
from crushed tissues 10 minutes in hot water (90°C., falling to 35°C.), squeezed dry 
in a hand press and filtered : 

Acidity + 180 (Fuller's scale). Xo reducing sugar present. It is a faintly 
clouded pink liquid. 

II. Juice of 9 fresh buds treated in the same way: 

Acidity +168. No reducing sugar. The pink liquid is decidedly cloudy and 
contains at least 10 times as much starch as I. 

C. I. In December, 1919, 24 rather slender shoots from side branches, each 
bearing several leaves, were cut away, weighed and left in a heap in an open 
wooden box on the hothouse bench fur three days, the first two of which were 
cloudy and wet: 

1. Fresh weight 242.25 grams. 

2. Loss of weight in 72 hours 59.75 grams. 

3. The 24 terminal buds were then removed, weighed, crushed (7 min.), ex- 
tracted 8 minutes in 50 c.c distilled water at 90°C. falling to 35' C, squeezed 
dry in a hand press, filtered and titrated. The fluid was a cloudy pink which a 
drop or two of the alkali changed to colorless: 

Acidity + 195 (Fuller's scale). 

II. Juice of 24 fresh buds treated in the same way: 

Acidity + 190. 



602 



BACTERIAL DISEASES OF PLANTS 




Fig. 440. — A. No. 10, second series, main axis. Leaf J not dwarfed but 
enormously proliferous and twisted, 4000 shoots present, nearly all on upper surface. 



miscellaneous: experimental teratosis 603 

a series of proliferous leaves and internodes, corresponding to a 
series of pottings or water-interruptions, separated from each 
other by leaves and internodes free or nearly free from such 
shoots. 

24. The normal upward movement of water from roots to 
shoots being thus interrupted, the water movement will now 
be from inner parts of the stem toward the surface and especially 
toward the leaves, and undoubtedly some leaves will transpire 
more than others, making at times an unequal drain upon the 
young tissues which will be manifested later by the development 
of an unequal number of adventive shoots. There will be also, 
as I have said, an abstraction of food which must move down- 
ward to repair the roots. That there is often a real shortage of 
food in the plant at this time is shown by the frequent stunting 
of the shocked and proliferous leaves. Often, when full grown, 
although by no means always, the shocked leaf is plainly smaller 
than the leaves below it or the ones above it (Figs. 430P, 432X, 
437M). Occasionally it is not }i or }i the size of the next 
leaf below it or the next leaf above it ; although it is generally 
full of adventive shoots, or pimply with their rudiments. But 
on the other hand, a very profound phyllomania may occur 
(many hundreds of shoots developing) on big leaves (Figs. 432^, 
440j, 4464) which exhibit no evidences of starvation. I take 
the starvation and the shock, therefore, to be two distinct things. 
The shock, in other words, I believe to be of short duration, like 
that due to the particles of acetic acid water or other substances 
which start the growth of intumescences on cauliflower leaves, 
like the crown-gall stimulus which determines the growth of 
adventive shoots in tumors on internodes of various plants, or 
like Bataillon's needle prick which starts the unfertilized frog's 
egg into growth,^ while the starvation, if there is any, extends 

^ Comptes Rendus des se. de I'Acad. des Sci., Paris. 1910, Tome 150, page 966. 
Confirmed by Jacques Loeb. Proc. Nat. Acad, of Sciences, vol. 2, 1916, p. 313. 

Contrast J with K the next leaf above it which is nearly free from shoots. Axis 
proliferous at R especially on the back side, but very much less so than the leaf J, 
or than other internodes shown on these plates. For center of the twisted pro- 
liferous leaf blade (not shown here) see next plate. Photographed October 1, 
1918. About 3"^ natural size. 

B. Cross-section of a petiole-trichome showing the origin of a shoot. X 80 



604 



BACTERIAL DISEASES OF PLANTS 




Fig. 441.— Central part (upper face) oi leaf J, of Fig. 440. Proliferation 
chiefly from the midribs. Photographed October 1, 1918. Part out of focus 
because not all in one plane. X 5, nearly. 



miscellaneous: experimental teratosis 



605 




Fig. 442.— Jso. 10, second series, middle leaf of a branch from the axil of leaf 
(r. Most shoots are near the main source of food and water supply, i.e., are in the 
vicmity of the midribs. Photographed October 1, 1918. X 4 circa 



606 



BACTERIAL DISEASES OF PLANTS 




Fig. 443. — No. 15, second series, main axis. Upper face of leaf L, •which was 
embryonic and stipule-wrapped on July 24. On the blade there are 1700 embryo 
shoots, mostly upon or near the main ribs; on the petiole, 420 shoots. Photo- 
graphed October 7, 1918. About H natural size. 



miscellaneous: experimental teratosis 607 

over a considerable period, one long enough to produce a dwarf- 
ing from which the leaf cannot recover. Such leaves look 
sickly and may fall early. Occasionally, when they are greatly 
dwarfed, although extremely pimply, they bear very few shoots, 
as if the latter could not develop for want of food. 

The prolification, I think, can hardly be brought about by 
starvation which we know may throw dwarfed plants of various 
kinds into premature or excessive blossoming, since it is common 
observation that in a great variety of woody plants in full and 
vigorous foliage there is enough food present in the roots and 
shoots to make an entire new" set of leaves in case the leaves have 
been destroyed wholesale by caterpillars or by frost, but hardly 
ever enough for a third set of leaves. Moreover, this begonia 
may be kept in a small pot for a very long time without starving 
it into phyllomania. There would appear, therefore, to be an 
abundance of food in these broad-leaved vigorous plants so that 
totipotent cells in the embryonic epidermis need not behave as 
if in the last stages of starvation, even granting that they would 
proliferate, if starved. 

25. That there is excessive loss of water from the shocked 
leaves and internodes would seem also to be indicated conclu- 
sively by the fact that very frequently corky patches are de- 
veloped on such organs, especially on the internodes (Figs. 
432, 4343, 437, 4493, and Table III), often entirely surrounding 
them, while cork is present nowhere else on the plant either above 
or below. This, I interpret, as a more or less futile effort on 
the part of the organ to protect itself from loss of water. 

In this connection Series VI is also very interesting. This 
experiment deals with 59 plants. It was begun on March 5, 
1919, and the principal dates are as follows: 

March 5. Cuttings made and left to dry on the hothouse 
bench. 

March 7. Cuttings bedded in sand. 

May 7. Rooted cuttings transplanted to 4 inch pots. 

June 2. Transplanted to 8 inch pots. 

The plants grew splendidly and were mostly unbranched and 
about 15 to 16 inches high on July 12 to 23 when the tops were 



608 



BACTERIAL DISEASES OF PLA^'TS 




Jeft to sh'o,v ZT "^T ''T '""'" >""■' "' *"= '"•""Ode and' rotated 'oO" to the 



miscellaneous: experimental teratosis 609 

cut away (upper 4 to 5 leaves) mostly for the titration experi- 
ments described on page 600. 

Result in Novemher: Dormant buds just under these tops 
have now pushed stunted small shoots (2 to 4 inches) and these 
are covered with cork, some almost entirel}-, others partiallj^, 
and where not very corky they have also pushed adventive 
shoots. The leaves are small (stunted) and many of them have 
fallen. These small upper side shoots most of which were un- 
developed, but some of which were an inch or so in length when 
the top was removed, are the only corky parts of the plants, and 
each of the 59 plants (all of which were cut back) shows this cork 
phenomenon to a very striking degree. Some of the plants also 
show cork-formation on the main axis immediately under the 
cut (the upper inch or so of the stub). These are the stems 
which were softest at the time the tops were removed, and which 
lost most water, as shown by their contracted and flattened 
shape. It is a striking confirmation of my idea that loss of water 
induces cork formation and the adventive shoots. 

26. Why loss of water should shock dormant cells into growth 
I have undertaken to discuss in the preceding chapters. It 
seems to me likely that the initial stimulus to all spring growth 
of land plants may be conditioned on gradual winter losses of 
water through stems and twigs. This would be more rapid in 
mild winters than in cold ones. When the removal of water 
from the dormant buds has reached a certain point then they 
will begin to grow. Etherized plants also push their shoots 
earlier than normal plants and in this case also we may suppose 
that there is excessive loss of water from cells of the buds through 
the temporarily paralyzed protoplasmic membranes. We should 
distinguish, I believe, between the initial shock or stimulus, the 
cause of the first cell-division of a dormant totipotent ceU, and 
the subsequent growth, which latter depends on renewed food- 
supply, water-supply, proper temperature, etc., and will proceed 
as a matter of course, once the initial dormancy has been 
overcome. 

27. The prolification usually is most pronounced on the upper 
surface of leaves and petioles, that is, it is negatively geotropic, 
but sometimes it occurs, although less freely, on their under- 

39 



610 



BACTERIAL DISEASES OF PLANTS 




MT^*^" *^*^ 



Fig. 445.— Plant No. 1, third series (dried cuttings) at the end of three months. 
Leaves Tp and X have fallen. Tp was the small top leaf when the cuttmg was 
made; X and Y were stipule-folded rudiments. Cork at Ck. Stipules at St; 
Y was a dwarfed leaf. Axis smooth above and below the stimulated part. 
Photographed October 15, 1918. X 2, nearly. 



miscellaneous: experimental teratosis 611 




IQIQ T 7 f. '.T'^ ''"'' °^ ^'"^^^ ^""^'^g^- Made March 15, 

1919 Translerred from sa^id bed to a 4-inch pot May 7. Repotted in an 8-inch 
pot June 2. Photographed June 28, 1919. ProHferous leaf (No. 4) not dwarfed 
Its blade was 9^i mches long. Leaf 5 was full grown. Leaf 3 was not full grown 
Leaf 1 at the top, just under the bud was quite small and Leaf 2 had a blade about 

Nn Tk ' i-^^f • . T ""^ i^''' ^'^'''' '''''' proliferous on their under surface. 
No. 5 bore 8o shoots. No. 4 bore 2500 shoots, and No. 3 bore 116 shoots 



612 BACTERIAL DISEASES OF PLANTS 

surface (Figs. 438, petiole, and 448^, blade). Very frequently 
the proliferation is abundant only over the main ribs of the leaf- 
blade or in their vicinity (Figs. 438, 441, 442, 443) but not always 
(Figs. 427, 433Z, and 4464.). Sometimes it is restricted to the 
apex of the shocked leaf. This apical development I have 
observed 5 or 6 times. On the internodes it may be strikingly 
one-sided (Figs. 429C, 435, 4365, 4440, quite uniformly distrib- 
uted (Fig. 429A, B), or more on the upper or lower end, the latter 
depending apparently on the condition of the neighboring in- 
ternode, i.e., on whether it is proliferous. Thus, frequently, I 
found one internode proHferous throughout, but only the apex 
of the next below it, or only the base of the one next above it 
(see Table II). When the phyllomania is one-sided, involving 
two internodes, the leaf midw^ay on the free side of the stem is 
itself free (Fig. 435^"), while the leaf next below it and the one 
next above it (those on the proliferous side of the internodes) 
are both proliferous. This I explain, as already suggested, by 
supposing more water to have been drawn from one side of the 
stem than from the other. On the nodes, the chief seat of pro- 
liferation is around the stipule-scars, which would seem to be 
places where the plant is least well protected. 

28. In addition to being stunted, the shocked leaf-blade may 
be variously twisted and distorted (Fig. 440j), owing to ex- 
cessive proliferation from its main ribs, the tissues being crowded 
downward, since most of the shoots are from the upper surface 
and they must have room to grow. In this way the ribs may 
become abnormally thick, corky and cracked open in many 
places, quite as if parasitized. 

29. The adventive leaves in this begonia are not mirror 
images of the parent but quite normal in orientation (except 
positively geotropic on under surfaces) and, except for fusions, 
only abnormal in number, size, shape and vitality, because they 
are crowded and starved. I have seen one case in which that 
part of the dorsal surface of a leaf-blade directly over the petiole 
was fused its whole length to the dorsal surface of another blade, 
the petioles also being fused, but the blade-wings free (Fig. 
448C) ; other fusions are common. 

The abnormalities (Fig. 452) observed on these adventitious 



miscellaneous: experimental teratosis 613 




Fig. U7.-A, B. Plant III (Table I). Upper .surtaee ol a leaf-blade showing 
numerous proliferations from the thickened red border of small knife wounds 
made March 30, 1918, when the leaf-blade was quite voung (less than 1 inch long). 
Most of the shoots are too smaU to be seen distinctly. The surrounding tissue 
IS quite free from shoots. Photographed May 1, 1918. X 5. 

C, D. Upper surface of a leaf-blade showing proliferations from margin of holes 
bored with a hot needle on May 14, when the leaf-blade was 1^ inches long. 
Photographed July 13, 1918. X 5. 



614 BACTERIAL DISEASES OF PLANTS 

Bo •:!& % . ^i^>i:^ 




Fig. 448. — A. Leaf showing origin of shoots from the margin of a sht wound 
on under surface of a midrib: ^i, side view; A2, vertical view. The red leaf-blade 



miscellaneous: experimental teratosis 615 

shoots relate to: (1) dwarfing or disappearance of parts, (2) dis- 
tortion or enlargement of parts, (3) duplication or fusion 
of parts. For example, we may have petiole absent, petiole 
very short, petiole twice the length of the blade, petioles fused; 
blades widened and cleft at apex (variously 2-lobed), sinus at 
base closed (Tropaeolum leaf), blades spatulate (petunia leaf), 
blades destitute of serratures or serratures reduced to mere 
undulations, blades deeply incised (grape leaf), blades with 
basal lobes widened so as to be triangular in outline (ivy leaf), 
blades abnormally short and broad, blades abnormally long and 
narrow (olive leaf), tissue mostly wanting on one side, whole 
blade abortive, basal obliquity absent, two blades partly fused, 
i.e., petiole fusion continued into the blade. 

30. The shock is something which either causes the develop- 
ment of totipotent cells, out of young peripheral unipotent or 
pluripotent cells (the cambium of the plant is not involved) 
or else causes great numbers of totipotent cells, already present 
in the epidermis chiefly in its appendages (hairs and glands) 
but dormant, to be shocked into division, after which, that is, 
when the plant has acquired a new root-system and general 
growth has recommenced, they continue to grow until they 
appear above the surface as young plants bedded in the tissues 
of the mother plant. 

31. That the plant requires only a moderate amount of 
water for its well-being is well-known to gardeners. This is 
indicated structurally both by its elaborate sub-epidermal 
storage vsystem (hypoderm) and by its paucity of transpiring 
organs. On the green stems there are no stomata, so far as I 
have been able to see, but only a few^ lenticels, and on the leaves 
there are fewer stomata than one would expect from their abun- 

was folded and only ^i inch long when slit. Leaf wounded March 30 (Ii of 
Table I). Photographed April 25. X 5. 

Bi, Bi. Effect of dw^arfing on production of shoots from the edges of wounds. 
Leaves from IV i and IV 2 (Table I). Leaves wounded March 30 when quite small. 
The large leaf developed 283 shoots of which 241 were in the vicinity of the wounds. 
The small dwarfed leaf developed only 8 shoots, all from the edges of the wounds. 
Photographed May 3, 1918. ^y natural size. 

C. Two leaves grown together along middle part of dorsum. Adventitious 
shoot from a leaf-blade. X 5. 



616 



BACTERIAL DISEASES OF PLANTS 




Fig. 449.— 1, 2, and 4. Petioles developing shoots from triclionies at x, x. 
X 8 circa. (5) Under surface of leaf showing rib trichomes developing shoots 
at X, X. (3) No. 6, first series, a detail from Fig. 432; much cork on the lower of 
the two stimulated internodes. 



miscellaneous: experimental teratosis 617 




Fig. 450.— 4, B, C. Three petioles showing origin of shoots from trichomes. 
At X, X, the trichome has begun to shrivel; at y it has thickened throughout; 
at z it has not. Normal trichomes scattered about. X 7. 



618 



BACTERIAL DISEASES OF PLANTS 







f -i 





Yio 451 —A. Cross-section of a leaf from No. 18, second series, showing 
superficial origin of a shoot. It arises from the colorless tissue (epidermis) above 
the palisade tissue (p) which is unbroken. As such a shoot grows it displaces F 



miscellaneous: experimental teratosis 619 

dance on other green plants. On the upper surface of the leaves 
I have not found any stomata and on the lower surface the aver- 
age is only about 20 per square millimeter, but in this respect it 
is not different from a half dozen other begonias I have examined. 
32. When new phenomena appear our first thought is to 
inquire whether there are any old and well-known phenomena 
which can be linked up with the new appearances and thus serve 
to explain them and also whether there are any other unsolved 
problems on which they themselves will serve to throw light. 
In this case one naturally thinks of root-pruning or bruising, 
sometimes used to throw sterile fruit trees into bearing; of the 
development of young plants from the leaf margins of Bryo- 
phyllum calycinum, which also occurs, so far as I have observed, 
only when the water-supply is interfered with, i.e., w^hen the 
leaves are severed or partly severed from the stem or when water 
is withheld from the roots, or is drawn away from lower leaves to 
more active upper leaves, but here, while the stimulus appears 
to be the same, we have to do not with pathological or semi- 
pathological phenomena, or with regeneration, as I understand 
the term, but only with the growth of preformed dormant buds 
located in unusual places but otherwise normal ; of prolepsis and 
prolification in peach trees attacked by peach yellows and peach 
rosette, where I have satisfied myself that there is premature 
death of a great many feeding roots, so that loss of water might 
exceed the intake, although the cause of this root-injury vremains 
to be determined; of regeneration in general in plants and 
animals where the response is directly from the wounded surface 
or the cells in its vicinity, as it is in this begonia when young 
leaves are wounded; of crown-gall embryomas, where the shock 
which causes the development of roots and shoots in the tumors 
is connected with the presence in the tissues of the products of 
Bacterium tumefaciens and the growth of the tumors in the 
vicinity of totipotent cells, which are also set growing; of intu- 

and occupies deeper parts of the leaf . From a stained serial section. For a 
much eariier stage see Fig. 453 at A'. X 55 circa. 

B. Cross-section of an internode showing superficial origin of the proliferous 
shoot. The phloem-xylem is a long distance below the part here shown and no 
vessels extended into it. Section photographed unstained in water. X 75 circa. 



620 



BACTERIAL DISEASES OF PLANTS 




Fig. 452. — Leaves from adventive shoots showing fusions and other abnor- 
malities referred to in the text. At x, y, are stem glands (enlarged) giving rise 
to shoots at their base. 



miscellaneous: experimental teratosis 621 




Fig. 453.— Cross-section of a leaf of Begonia phyllomaniaca showing early 
stages of adventive shoots at x and y. The dark central band is the only part of 
the leaf producing chlorophyll. From series V at end of three weeks Photo- 
graphed m water from a thick free-hand section. 8 mm., 4 oc, bellows at 35 



622 BACTERIAL DISEASES OF PLANTS 

mescences due to chemical and mechanical injury or to frost 
where clearly there is always some initial loss of water and in 
some instances, at least, increase of acidity; and finally, of tera- 
tomas in animals where nothing is known as to cause but where 
the consensus of expert opinion appears to be that the totipotent 
or pluripotent cell or cells giving rise to the fetal fragments dates 
from embryonic time, and in case of the atypical forms although 
"out of place," would have continued dormant but for the shock 
of the developing cancer. 

33. In this connection one thinks also of the various processes 
used by gardeners to hasten the pushing of dormant buds. 

In 1885 Dr. Hermann Miiller-Thurgau showed conclusively 
that potato tubers of varieties which ordinarily do not sprout 
until spring can be made to germinate in autumn or early winter 
by exposing them on ice for a number of weeks. If they are 
then removed and placed under conditions suitable for growth 
the dormant buds immediately begin to grow. He showed that 
potato tubers placed under these conditions change a portion of 
their starch into sugar and he believed that this increase of 
sugar is the cause of the germination. He mentioned incident- 
ally that there is also an increase of acid but lays no stress on 
this increase which, however, I believe to be the actual cause. 
His chilled potatoes which had become sweet were twice as acid 
as the unchilled ones (3.14 pro mille reckoned as malic acid, as 
against 1.74). 

In 1900 Dr. W. Johannsen, the Danish physiologist, experi- 
menting at first with sulphuric ether discovered that a great 
variety of plants which ordinarily do not push their winter 
buds until spring can be induced to push them in late autumn 
or early winter by etherizing the plants or by chloroforming 
them. Lilacs, for example, by this method can be brought into 
blossom at Christmas time and willows can be induced to push 
their catkins in autumn within a week or ten days of the time 
they have been anesthetized. This method of procedure, 
worked out in detail by Johannsen for a variety of plants, proved 
so dependable and profitable that it is now used by florists the 
world over. The dose, temperature and time for lilacs is as 
follows: 30 to 40 grams of ether per hectolitre of air space 



miscellaneous: experimental teratosis 623 

(about 3)2 cubic feet), a temperature of 9'' to 20°C., and gener- 
ally an exposure of 48 hours. When chloroform is used the 
time and temperature may be the same but the dose is reduced 
to 6 to 9 grams per hectolitre. The soil must be fairly dry 
otherwise much ether will be absorbed and the results dis- 
appointing. Ether vapor being heavier than air and explosive 
in the presence of fire, it must be liberated in the top of the air- 
chamber and the work must be done out of door or in a room 
where there is no fire. Johannsen states that he derived much 
benefit from a perusal of Miiller-Thurgau's writings and from 
the earlier work of the great French physiologist, Claude Bernard. 

In 1909 Dr. Hans Molisch of Vienna published a very inter- 
esting paper showing that the same results obtained with ether 
and chloroform can be obtained simply by dipping the tops of 
the plants into warm water (30° to 40°C.) for a short period 
(6 to 12 hours). They are then set on the greenhouse bench 
under suitable conditions and bloom prematurely just as if 
they had been etherized. This method he states had been used 
for a considerable time b}^ Russian gardeners but Molisch was 
the first to make exact experiments and to bring it to the 
attention of the scientific world. 

Neither Miiller-Thurgau, Johannsen nor IMolisch have off- 
ered satisfactory^ explanations for the results obtained. Miiller- 
Thurgau's explanation that the hasty pushing of the buds is due 
to the presence of sugar in the tissues cannot be held to be a 
proper explanation since, as Johannsen points out, plants in a 
dormant state sometimes contain sugar in quantity, for exam- 
ple, garlic bulbs, and there is also always a considerable quan- 
tity of sugar circulating during the summer season in a variety of 
deciduous plants while their winter buds are forming and yet 
these buds do not ordinarily push. Preceding Johannsen's 
work Dr. Raphael Dubois, professor of physiology in Lyons, 
France, published a paper (1891) on the action of chloroform in 
which he claims that the anesthetic acts by dehydration. I 
was unaware of the existence of Dubois' paper until November, 
1919, when I read for the first time Johannsen's paper, although 
I had already arrived theoreticallj^ at the same conclusion, as 
may be seen from the preceding pages which were then in type. 



624 BACTERIAL DISEASES OF PLANTS 

Johannsen makes light of the work of Dubois, as does also 
Overton. Johannsen objects that the figure which Dubois 
shows (that of an Echeveria exposed to chloroform and 
exuding drops of water from all of its leaves) is the figure of a 
dead plant, and this may well be, but clearly there must be all 
stages in the exudation of cell-water from its mere beginnings in 
the live plant to its end, when not only the intercellular spaces 
and the sub-stomatal chambers are filled with the exuded sap, 
but also a sufficient quantity has exuded to appear on the sur- 
face in the form of tiny drops and to have exhausted the cells 
beyond recovery. In fact, Overton admits that there is loss 
of water from narcotized muscle and that Dubois is not wholly 
wrong. Recently I have observed all stages of dehydration in 
chloroformed cabbages. If the anesthetization is moderate 
there is only a spotting of the leaves (innumerable tiny spots, 
water-soaked or darker green by reflected light and translucent 
by transmitted light) without surface exudate. If the chloro- 
forming is continued longer, the change of color overspreads 
the whole leaf, and, if it is an undeveloped leaf with small inter- 
cellular spaces, there is an exudate of clear fluid from hundreds 
of stomata mostly to the under surface of the leaf, but, if it is 
an older leaf, the large amount of intercellular space is sufficient 
to accommodate the exuded water and it seldom appears on 
the surface. What becomes of such leaves depends on how far 
the dehydration is pushed. If the experiment is stopped before 
the exudate or spotting appears the leaves recover, if pushed 
until exudate appears on their surface I have not seen them 
recover. 

Temporary loss of function on the part of the protoplasmic 
cell membrane whether brought about by chilling, by hot water, 
by anesthetics, by acids, or by alkalies would lead, I believe, to 
the same set of phenomena; viz., loss of water, disturbed respira- 
tion, more or less oxygen-hunger, and compensatory cell-division 
with movement of water, sugar and other foods back into the 
growing point. Once the dormancy is overcome buds will 
continue to grow if placed under growing conditions. I believe, 
therefore, that we have in these common forcing methods added 
evidence that the phyllomania in this begonia must be due to 



miscellaneous: experimental teratosis 625 

the shock produced by excessive loss of water acting on dormant 
totipotent cells. 

34. I am inclined to think there is nothing in Weissmann's 
theory making a sharp distinction between somatic and germinal 
cells. I believe that cells in many young undifferentiated parts 
are totipotent and that what finally becomes of them, that 
is whether they avail themselves of their totipotency, or not, 
depends on circumstances. Under ordinary conditions we know 
how they behave (that is, physiologically) but under extraordinary 
conditions they may behave quite differently, that is may at- 
tempt to reproduce the whole organism. In this begonia, toti- 
potent cells are distributed in great numbers over its superficial, 
actively growing parts. They occur, I believe, similarly if not 
so abundantly in the epidermis of other plants and in the skins 
of animals, but in most cases require a much stronger shock to 
set them growing, that is, a tumor, or a parasitic stimulus of 
some sort. That pluripotent cells should occur in the skin of 
a man, let us say, seems a very strange thing, yet man has in- 
herited his skin and all the rest of his anatomy from the lower 
animals, some of which, for aught we know, may have germinal 
cells as widely distributed and as sensitive to shock as they are in 
this curious begonia. No one knows, for instance, what effect 
a shock of some sort, such as a severe blood-letting or a drastic 
purgation of the mother, might have on a fetus in the way of 
starting dermoid cysts or other monstrous growths. The subject 
is one calculated to provoke thought and lead to further experi- 
ments. The effects of root-injury on this plant seem to me very 
suggestive as to the origin of Sereh of sugar-cane, of rosette 
diseases, of orange wilt, of peach yellows, and of the somewhat 
similar East Indian spike disease of sandalwood. It has also 
various other interesting bearings. 

35. That this begonia is more subject to shocks leading to 
phyllomania than other plants may be conceived to be due to its 
watery nature and especially to its inheritance of very sensitive 
and easily permeable cell-membranes. That it is alone in the 
world, in such behavior, I do not for a moment beheve. 



40 



626 



BACTERIAL DISEASES OF PLANTS 



TABLE I 

Showing result op wounding undeveloped leaf blades of Begonia phyll- 
omaniaca. Knife wounds made March 30, 1918. Examinations made 

AFTER a month 



Plants and 
branches 
of plants 



69 unwounded vs. 
13 wounded leaves 



Average 
number of 
shoots per 
sq. in. on 
unwounded 
leaf blades 
Total 

surface, 
784 sq. in. 



Average 
number of 
shoots per 
sq. in. on 
wounded 
leaf blades 



Contrasts on wounded leaves 



Wounded parts 



Fraction 
of sur- 
face oc- 
cupied 

by 
wounds 



Total 
number 

of 
shoots 
(com- 
pare 
with 
last col- 
umn) 



Shoots 
per each 
\i sq. in. 

of 
wounded 

surface 



Unwounded parts 



Shoots 




per 


Total 


each 


sur- 


J-8sq. 


face 


in. 





Total 
shoots 



Average 
number of 
shoots on 
any equal 
fraction 
(For comp. 
with col- 
umns 4 
and 5) 



III lower leaf 
III upper leaf 
112 

Ill, 

III2 

III3 

IVi 

IV2 (dwarf 
shoot) 

Vi lower leaf. 

Vi upper leaf . 

V2 

Av. 5 plants 

(11 branches) 



2.0 
0.61 



1.0 
1.6 



0.0 
1.3 



0.33 



1.2 



17.3 
19.0 
9.8 
11.6 
20.6 
21.0 
19.0 
36 
15.0 

0.27 
6.7 
14 
8.0 

15.2 



K2 

Hs 
Ml 

Ml 

H 

H9 

Me 
Ha 



215 
183 
71 
117 
238 
141 
246 
267 
241 



46 
145 

58 

152 



61 
32 
43 
24 

47 
49 
89 
48 

3 
31 
24 
39 






07 





125 





5 





09 





2 





18 





3 











4 





24 





04 



1H2 

'Ho 

1%9 



'%9 



'Hi 



46 
8 
86 
57 
51 
5 
24 
11 
42 


41 
22 

3 



0.55 
1.5 

0.0 
0.6 
1 .5 
07 

1.4 



' There was also a leaf (I3) which received eleven wounds, but the total wounded surface was 
not recorded. This was on a dwarfed shoot from the axil of the branch I2 and there were no shoots 
either from the wounded or unwounded parts. 



miscellaneous: experimental teratosis 



627 



TABLE II 

Showing number of shoots on internodes stimulated July 24-25, 1918, and 
ON unstimulated internodes immediately .vbove and below the same. 
Counts made in September-October, 1918 



Series 

and 

plant 

number 



Part used 



Number of adventive shoots on internodes 



Internodes next below 



Stimulated 
internodes 



Internodes 
next above 



lis 
He 

Il7 

He 
IIii 

IIl4 
IIl5 



Main axis. . 
Main axis. , 
1st branch. 
Main axis. . 
Main axis. , 



Underground branch 

3d branch 

3d branch 

6th branch 



1st branch. 
3d branch. 
Main axis. 



1st branch (arising 

underground) .... 

4th branch (K) . . . 

6th branch 

7th branch 

Main axis 

Main axis 



5 





9 









2 


1 






6 







44 
5 


10 



16 inches practically free 





20 

1 



3 

1 

25 






















10 



5 


9 


8 


7 





6 


5 


5 













Branch E. . 1 

Main axis jOOO 

1st branch 0000 

2d branch 000 

Main axis 3 4 

4th branch 

Main axis 03 5 

Main axis 0000 

Main axis 2 21 

Main axis 22 

2d branch 00 

3d branch 14 

Main axis 2 16 

top 
Main axis | 2 1 301 

top 
(one side) 

Main axis 130 shoots in a length of 

8 in. 



26.5 1150 

235 18 

220 
295 130 

Not counted but 
very proliferous 
360 700 

300 



134 
18 
(all at top) 
160 
156 
850 



3d branch (F), 











16 
280 

130 

195 

40 

(all on one 

side) 



360 



173 
200 



126 

285 



62 
95 



215 



140 
95 



65 



110 



42 



160 

18 



lO! 

3 

6 3 

4i 2 







2i 2 

5 3 



(free except 
at base) 

105 
22 (base) 
480t 

32 
250 



65 



.540 
180 



170 



28 



160 
300 



220 



3 
5! 10 
3 2 

0| 
3j 2 
0' 





o| 



3! 



(corky) 

33 95 
(very corky) 



675 



110 



30 
(free except 
base) 
35 



01 

3} 5 

4: 1 

101 

36 2 



0| 

o| 









4 5 





4 






Oi 

4 






12 



1 







10 on 4 inter- 
nodes 



r 



* No internodes below first recorded one. 

t When but a single number is given there was only one proliferous internode. 

t In cases like this the shock undoubtedly influenced more than two internodes. 



628 



BACTERIAL DISEASES OF PLANTS 



i§ 2 




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< O Q 




5! c3 12: 




^ ^ n 




^ H 












-- fe ^ 










-r, '^ Z 

w = -< 

£ J ss 






2 ^ "^ 




2 < a 




s t3 a 




S § z 




CO 


o g o 


9 ^ «= 
z p a 


^ 


a H > 


z 


5gS 


< 


p 


,— , H 




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miscellaneous: experimental teratosis 629 

begonia literature 

1852. Begonia phyUomaniaca Martius. Hooker's Journal 
of Botany, Vol. IV, pp. 206-207. 

1853. De Martius, C. F. Ph. Begonia phyUomaniaca in 
''Delectus Seminum in Horto R. Bot. Monacensi" Anno 1852 
Collectorum. Annales des Sciences Naturelles. 3 Serie, 19, 
1853, p. 366. 

1854. Von Martius. Flora Brasiliensis, Vol. IV, Pars I, 
pp. 386-387. Plates XCIX and C. 

1854. Klotzsch, Hrn. Begonia phyUomaniaca in 
''Begoniaceen-Gattungen imd Arten,'' Abhandlungen der 
Koniglichen Akad. der Wissenschaften zu Berlin, 1854, p. 129. 

1863. Verlot, J. B. Begonia. Bulletin de la Societe 
Botanique de France. Tome X, p. 474. 

Eight lines from a letter in which he states that hairs on the leaves of a begonia 
(sp. called B. geranioides) are changed into buds which render the leaves in some 
sort viviparous. The letter and specimen were sent to M. de Schoenefeld who 
exhibited them at a meeting of the Societe Botanique de France. 

1863. Prilleux, Ed. Observations sur une feuille gem- 
mipare de Begonia. Bulletin de la Societe Botanique de France, 
Tome X, pp. 492-494. 

At a following meeting of the Societe Botanique Prillieux discussed Verlot's 
discovery and fogged the whole subject, denying that the shoots were the out- 
growth of hairs. 

1875. Caruel, T. Nota su di una trasformazione di 
peli in gernme. {Begonia phyUomaniaca.) Nuova Giornale 
Botanico Italiana, Vol. 7, pp. 292-294, Pisa, 1875. 

Note on plants seen at the Kew Gardens in London. His observations confirm 
Verlot's statements that hairs are converted into buds. 

1880. Hansen, Adolph. Vergleichende Untersuchungen 
iiber Adventivbildungen bei den Pflanzen. Abhandlungen 
Herausgegeben von der Senckenbergischen Naturforschenden 
Gesellschaft. Vol. XII. Frankfurt, pp. 147-154. 

1887. DucHARTRE, P. Sur un Begonia phyllomane. Bul- 
letin Societe Botanique de France. Tome XXXIV, Paris, 1887, 
pp. 182-184. 

Forty plants raised by Nodot from seeds showed phyllomania on their stems. 



630 BACTERIAL DISEASES OF PLANTS 

One of these was studied by Duchartre, who says, "How are these little supple- 
mentary leaves produced? I have had neither the time nor the material neces- 
sary for seeking their first origin. . . . Each one of these growths constitutes in 
reality a leafy branch" 

As a simple hypothesis he thought they originated from the epidermis. 

1894. Warburg, O. Begonia phyllomaniaca. Die natiir- 
lichen Pflanzenfamilien. Engler und Prantl. Teil III, Abt. 6 and 
6". p. 124. Leipzig. 

1908. GoEBEL, K. Einleitung in die Experimentelle 
Morphologic der Pflanzen. Naturwissenschaft und Teehnik 
in Lehre und Forschung eine Sammlung von Lehr- und Hand- 
biichern. [Begonia phyllojiianiaca on pp. 153-155.] 

1913. Bateson, William. Problems of Genetics. [Begonia 
phyllomaniaca on pp. 50-53.] Yale University Press. 

1917. Smith, Erwin F. Embryomas in Plants (Produced 
by Bacterial Inoculations). The Johns Hopkins Hospital 
Bulletin, Vol. XXVIII, September, 1917. Also a repaged 
separate. Foot note on page 279. 

1919. Smith, Erwin F. The Cause of Prohferation in 
Begonia phyllomaniaca. Proc. Nat. Acad. Sciences, Vol. V, 
February, 1919, pp. 36-37. 

literature on effect of cold, heat and anesthetics 

1885. Miiller-Thurgau, Hermann. Beitrag zur Erklarung 
der Ruheperioden der Pflanzen. Landw. Jahrbiicher. XIV 
Bd. Berhn. Paul Parey, 1885, pp. 851-907. 

1891. Dubois, Raphael. Mecanisme de L'Action des Anes- 
thesiques. Revue Generale des Sciences Pures et Appliquees. 
Tome 2, Paris, Sept. 15, 1891, pp. 561-567. 2 text Figs. 

1900. Johannsen, W. Mein Aetherverfahren in der Praxis. 
Die Gartenwelt, 5. Jahrg., 1900-1901, p. 265. 

1901. Overton, E. Studien iiber die Narkose zugleich ein 
Beitrag zur allgemeinen Pharmakologie, pp. 195. Jena. Ver- 
lag von Gustave Fischer, 1901. (Dr. E. Overton, Privatdocent 
der Biologic und Assistent der Botanik an der Universitat 
Zurich.) 

1904. Johannsen, W. Friihtreibversuche mit Strauchern 
nach erfolgter Aetherisierung oder Chloroformierung. Sitzgs- 
ber. der "Flora/' 1902-1903. Dresden 1904, pp. 71-83. 



miscellaneous: experimental teratosis 631 

1906. Johannsen, W. Das Aether verfahren beim Friihtrei- 
ben, mit besonderer Beriicksichtigung der Fliedertreiberei. 2 
Aufl., pp. 65. Jena. Gustav Fischer, 1906. 

1909. Molisch, Hans. Das Warmbad als Mittel zum 
Treiben der Pflanzen. Jena. Verlag von Gustav Fischer. 1909, 
pp. IV, 38. Mit 12 Figuren im Text. 

additional literature on intumescences 
(See page 572) 

1898. TuBEUF, C. VON. Uber Lenticellen-Wucherungen 
(Aerenchym) an Holzewiichsen. Forstlich-7iaturwissenschaftliche 
Zeitschrift, VII Jahrgang, 12 Heft, December, 1898, pp. 405- 
414, 7 Figs. 

This very interesting paper, which was read with fear and trembling, came 
to m.v knowledge too late to be mentioned in the body of the text (page 477 
et seq.), i.e., not until the corrected proofs had been returned to the printer. It 
anticipates by twenty years some of my findings but not all. The author 
raises the question of reduced transpiration as a possible cause of the tumors 
and decides against it because he also obtained them in his closed vessels when 
projecting leaves were on the plant and transpiration was in progress, but if the 
fluid transpired possessed a limited oxygen-content as it must in closed tubes, 
the peripheral stem tissues would still receive insufficient oxygen; and they 
might even, if the water transpired by the leaves, had sufficient oxygen, i.e., 
was ground water, since the moist air in contact with the lenticels in the closed 
chambers would soon become deficient in oxygen. Concerning increased acidity 
of such tissues it does not appear to have occurred to Dr. Tubeuf to make 
any inquiries. 

Von Tubeuf states that the pi'oliferation may be so extreme as to be patho- 
logical and that it is not due to an excess of water in the tissues. He cites 
Goebel to the effect that it arises in consequence of an undetermined irritation 
and Schenk that " it is not very probable that simple contact of the epidermis 
with the water, as such, is a factor, it is much rather to be supposed that lack 
of oxygen in the inner tissues, the plasma of the phellogen cells, leads to the 
production of the aerenchym." 

Dr. Tubeuf's experiments were made with stems and roots of small trees — 
elms, etc. 

1913. Wehmer, C. Ubergang alterer Vegetationem von 
Aspergillus fumigatus in " Riesenzellen " unter Wirkung ange- 
haufter Saure. Berichte der Deutschen Botanischen Gesellschaft, 
Vol. XXXI, No. 5, 1913-1914, pp. 257-274, 7 text figures. 

Wehmer obtained great numbers of giant cells in cultures of Aspergillus and 
Penicillium, using ammonium sulphate. He attributes these giant cells to the 
action of free acid ions on the spores of the fungus. 



632 BACTERIAL DISEASES OF PLANTS 

1919. Taylor, William Randolph. On the production of 
new cell formations in plants. Contributions from the Botani- 
cal Laboratory of the Univ. of Pennsylvania, Vol. IV, No. 2, 
pp. 271-299, 8 pis. 

1920. RuMBOLD, Caroline. The injection of chemicals 
into chestnut trees. American Journal of Botany, Vol. VII, 
No. 1, Jan., pp. 1-20, 7 text figures. 

1920. RuMBOLD, Caroline. Causes for the production of 
pathological xylem in the injected trunks of chestnut trees. 
Phytopathology, Vol. X, No. 1, Jan., pp. 23-33, 2 pis. 

1920. Harvey, R. B. Relation of Catalase, Oxidase, and 
H+ Concentration to the Formation of Overgrowths. Am. 
Journal of Botany, May, 1920, pp. 211-221. 



PART V 
GENERAL OBSERVATIONS 

How to make the most of one's education, how to achieve 
the largest success, must ever be a matter of immediate concern 
to the student w^ho has to win his own way. With such persons 
in view, and I am speaking to no others in these pages, I will 
here set down some observations that have grown out of my 
own experience. If occasionally they prove useful and help to 
smooth ways which are often hard in the beginning, I shall 
feel well repaid. I have expressed my individuality very de- 
cidedly on a variety of subjects in the following pages but I 
could not do otherwise. If anyone thinks these observations 
smack too much of "Thus spake Zarathustra" he has the remedy 
in his own hands. We are often compelled to listen to an in- 
dividual when we are bored, but never to a book. '^ Si ce livre 
me fasche, i'en prens un aultre^ 

ON SUBSIDIARY STUDIES 

I have spoken farther along about the need of modern lan- 
guages and may say a word here about the despised Latin and 
Greek. As cultural studies, there can be no doubt of their value. 
The student of Latin and Greek is generally a more discrimi- 
nating student and forceful writer of his own language than other 
men and this is a sufficient reason for their study. In the case 
of the naturalist there are other reasons: (1) the terminology 
of science is derived from these languages, and (2) all the oldest 
scientific writings and some of the modern ones are in Latin 
and Greek, and these, in some instances at least, must be read. 
Finally, Latin is the mother of all the great Romance languages, 
whose literatures will be to you a source of profit and delight 
for many other reasons than the purely pathological one. My 
advice to the pathologist therefore would be: study both Latin 
and Greek, or at least Latin, and get as much out of it as you can. 

633 



634 BACTERIAL DISEASES OF PLANTS 

Of the sciences, the higher mathematics would seem to be 
of least use to the experimental pathologist, and yet I maj^ be 
wrong in this judgment . Certainly the end of all experimenting is 
to be able to express one's data in plots and curves, but biology 
is a very complex subject, too complex apparently for any mathe- 
matician to understand, and biologists, for the most part, are 
very far from being able to express themselves after the manner 
of mathematicians, however desirable it might be. Their 
language and ours are unlike almost to mutual exclusion. If, 
then, you are onl}^ an average biologist do not spend several 
years on the higher mathematics, because in the end you will 
be only an indifferent mathematician, a plodder and a grubber 
like the rest of us, not a member of the great race. When, 
as a student, I lamented to Harrington, the astronomer, my lack 
of proficiency in the higher mathematics, he said: "You have 
not cut as much underbrush in this direction, that is all." But 
I am sure the defect lies deeper, viz., in a tj^pe of mind, and one 
very common among biologists. The case is quite different, 
however, if your liking for mathematics is second only to your 
love of biology. Then you maj^ study it as long as you feel 
inclined. You will be a kind of a white blackbird among your 
fellow biologists but this need not disturb you, since you will be 
able to do some things which they cannot do. 

Of sciences which are closer to the pathologist I may mention 
experimental physics (especially those branches of it dealing with 
heat, electricity, hydrostatics, surface tension, viscosity, etc.) 
and chemistry, of which he cannot have too much. Bio-chemis- 
try in particular will be of service to him at eveiy turn. He 
cannot do without it unless he can arrange to work jointly with 
some chemist and even then he should not be content simply to 
look over the fence. The tj^pe of chemistry the pathologist 
should cultivate is that which deals with organic compounds 
such as his parasites produce or attack, and the problems con- 
nected with which he will have to face. I mean the chemistry 
of starches, sugars, celluloses, pectoses, tannins, acids, aldehyds, 
amino acids, glucosides, enzymes, ethers, esters, and the like. 
The pathology of the future lies right in the midst of these things 
and more and more the pathologist must be a chemist if he 
would succeed in a large way. 



GENERAL OBSERVATIONS: ON SUBSIDIARY STUDIES 635 

The student should also know something of meteorology 
and of surface geology and soil physics. He must have some 
knowledge of zoology and especially of entomology, both because 
insects act as carriers of disease and because he must know how 
to keep his experimental plants free from all sorts of depreda- 
tors. He should certainly know all the common insect pests, and 
the broad general conditions under which all animal life develops 
and functions. To know these things will give him a much 
broader and firmer gi-asp of his own problems. 

In botany, the pathologist may be trusted to acquire as he 
goes along a knowledge of the morphology and structure of 
plants because all his life he will be making sections of various 
organs on a variety of plants, but plant physiology he should 
study thoroughly from the beginning, for how can one know the 
meaning of a disease if he does not know the functions and 
behavior of a normal plant ! He should also understand garden- 
ing, that is the proper care and cultivation of plants in the open 
and under glass, and to this end he should affiliate with compe- 
tent gardeners. 

There is only one other group of studies I would touch 
upon. Human and animal pathology and modern medicine, 
with its stimulating outlook, are close neighbors, and the plant 
pathologist will be wise to make friends with the well-trained 
physician and the animal pathologist and to keep in touch as 
much as he can with the progress of these sister sciences. 

There is a large program laid out, I hear it said. So be it, 
but if you are not lazy nor wasteful of your time, but hew to 
the line through a series of years you can accomplish it all and 
much more, and tnust, because what I have mentioned is only 
subsidiary to the main task. 

• ON SEEING THINGS 

The successful student of nature, and especially the successful 
scientific man, must not belong to that type against whom it was 
said of old ''Having eyes they see not!" In him ''that inner 
eye which," according to the poet, ''is the bliss of solitude" 
must be forever open to the faintest impressions from the 



636 BACTERIAL DISEASES OF PLANTS 

natural world, if he would fathom its meaning. Seeing is not 
enough but it is the first step, the beginning of all the others. 
How to see with the eyes of a Darwin, a Pasteur, or an Asa Gray, 
that is the question! Poets are said to be born, not made, and 
inheritance must also play no inconsiderable part in the lives of 
all great men of science. Yet another saying has it that genius 
counts only for one-tenth while hard work is nine-tenths of 
every man's success. These are extreme statements and the 
truth lies somewhere between the two. Both environment 
and heredity are important. Certain it is, however, given some 
basis of good material to work upon, that patience and perse- 
verance will do much to cultivate and sharpen the seeing eye. 
This must be so, otherwise the amateur would be as efficient 
as the highly trained man and we know that this is not the case 
in any field of endeavor. As every teacher knows, it is hopeless 
to try to make students out of many persons because ''It isn't 
in them," as the saying goes. They carry an insurmountable 
inheritance of dullness. On the contrary, long pondering on a 
subject with oft-repeated observations of the physical phe- 
nomena involved gradually enables the right sort of a person to see 
definite principles quickly and clearly in that which was at first 
only a maze of obscurity and uncertainty. The plainest things 
are often the hardest to see because all our seeing and all our 
thinking runs, or is apt to run, in stereotyped channels and the 
older we grow the greater the danger. Strive, then, to keep an 
open mind and to enlarge your horizon as you grow old. 

But the first inertia is the most difficult to overcome — 
c'est le premier pas qui coute. The new and strange are always 
hard to comprehend and interpret. For this reason the first 
foreign language, especially if it is Latin or some other much 
inflected tongue, is I believe, always hardest for English-speak- 
ing persons. A Chinese student once told me that Latin was 
easy for him (because inflected, I presume) but English "very 
hard." For the same reason first impressions of a strange coun- 
try are always most vivid but generally very inaccurate, witness 
many books. Every one has heard the story of Agassiz's stud- 
ent to whom a fish was given that he might point out its most 
conspicuous feature. The bilateral symmetry of the fish was 



GENERAL OBSERVATIONS: ON SEEING THINGS 637 

what the master had in mind, but this was the very last thing the 
student thought of.^ Why? 

Learn then to see, and to think upon what you have seen! 
And look again and again lest you should miss something. 

By seeing I mean not loose general observations such as 
would enable you to distinguish a man from a tree (Smile, if you 
will, but this is the common way of seeing. I have exaggerated 
it only a little) but patient, long-continued discriminating 
observation. In this way, gradually, all the hidden details of an 
object become visible. When they are clear enough to be drawn 
or to be reflected upon as separate entities then only can you 
be said to know them. By thinking I mean prolonged log- 
ical reflection leading to clarity of view, not mere hap-hazard 
dreaming. 

"Learning without thought is labor lost; thought without 
learning is perilous." 

ON EXPERIMENTATION 

Observations and reflections, however extensive and pro- 
found, are not sufficient guides in pathology. These might serve 
to make a statesman or a philosopher but not a scientist. 
Things observed are to be questioned — and this questioning is 
done by means of well-planned experiments. These experi- 
ments lead necessarily to many new observations and often to a 
materially changed point of view, so that the imperfect frame- 
work of a discovery, which may have been nine-tenths insight 
at first, is gradually filled in and worked over experimentally 
until it becomes a substantial and lasting structure. In path- 
ology, as in all subjects dealing with phenomena, experimental 
tests of the validity of one's ideas are necessary at every step 
and the term '^scientist" is a misnomer when applied to any 
one who does not try his hypotheses in the reducing fire of 
experiment. The world is full of shouting theorists who have 
never made an experiment in all their lives, certainly not 
one worthy of the name, and yet they are asking all men to 
follow them. This is why most politics, economics, socialism, 
spiritualism, psychic research, psychology, philosophy and 

1 Doubtless, Louis Agassiz (1807-1873) tried this on many students, but Dr. 
W. J. Beal is the one who told it to me. 



638 BACTERIAL DISEASES OF PLANTS 

theology are such bogs and quicksands of the human intellect. 
The}' have not been, and from their very nature, in many cases, 
cannot be subjected to rigid experiment, and, therefore, have 
not arrived at, and in many cases, never can arrive at cer- 
tainty. They belong on another plane, that of possibilities or 
probabilities and some are not even possibilities. 

The best advice I can give the young pathologist is this: 
// you would go far, experiment continually. Try out all of 3'our 
theories and other men's theories by experiment. Let no day 
pass without something done to verify the correctness of the 
various ideas you have formed from your observations. In 
this way you will be able to discard many specious but erroneous 
assumptions, and will be continually adding to your sum of exact 
information. 

The reason many men are only hewers of wood and drawers of 
water is because they are content with simple observations and 
reflections, often very superficial ones, and stop short of experiment 
which would show them where the truth lies. They may lack 
the seeing eye and the inquiring mind, may have ''hook worm," 
be simply lazy, or perhaps only untaught. In too many children 
the eager questioning spirit is repressed by a hard and unsympa- 
thetic environment. Such persons are conspicuously weak in 
memory, and in a knowledge of the past. Consequently they 
are the natural and easy pre\' of the walking delegate, the 
political demagogue and the yellow journalist. 

ON BEGINNING WORK THOUGHTLESSLY 

The best ad\dce I can give the ambitious student is this: 
As far as possible, think out carefully in advance all the main 
ramifications of your experiments. This is not easy, even for 
the advanced worker, and surely you will have overlooked 
something, however thoughtful you may be, but by such 
preliminary cogitation you will escape many pitfalls, and come 
at once into the only proper way of research. "But I cannot 
afford the time," I hear someone say. Well, time wisely spent 
in the beginning of an undertaking is often time saved in the end. 
The shrewd commander takes into account all possible contingen- 



GENERAL OBSERVATIOXS : BEGIXXIXG WORK THOUGHTLESSLY 639 

cies, as nearly as may be, and thereby wins a battle or a cam- 
paign. The commander who cannot afford the time, or who 
lacks the foresight and the acumen, is beaten and disgraced. 
You have your choice. But what profit a student thinks he 
will derive from a blundering course of experimentation ending in 
some dead end or no thoroughfare, I cannot imagine. Your 
results, you may be sure, will not be commensurate with your 
labors. '^Palma non sine pulvere/' Yes, but Seneca is careful 
to add "per mam rectam. " You may flounder through the mud 
and dust desperately, but if you are on the wrong road all of your 
energy will not save \o\x. 

Literature is full of examples of this sort of bungling, espe- 
cially Theses, which once printed have to be read but which realh' 
have no raison d'etre, since often they do not add materially 
either to human knowledge or to the reputation of the writers. 
Sit down, therefore, with your problem and think it over 
seriously in all its various aspects before you attempt a stroke of 
work. The more thought you put upon it in advance the more 
likely you will be to obtain convincing results when you actually 
begin to experiment. Here I cannot resist telling an old story. 
An Irishman invented a cumbrous cover to keep water out of a 
gig in case of sudden rains while on the road, which cover, he said, 
was to be stored away underneath the gig in clear weather. 
''But there is no room for it underneath," said a critic. As this 
was only too evident, Pat was nonplussed for a moment and then 
rephed, "Well, you can leave it at home."" This is like many a 
human cogitation! Tried out it does not work! 

I am not attacking any one. Some of my own experiments 
have been of this sort, but fewer I trust in recent years than 
earlier. Now I always spend more time, often very much more, 
thinking over my proposed experiments, than I do in the per- 
formance of them. And generally speaking. I know in advance, 
barring some unforeseen contingency, just how they are coming 
out. If they fail, I begin to search shamefacedly for that 
something which I have overlooked, and sometimes it turns out, 
when discovered, to be as plain as the nose on a man's face, or as 
the bilateral symmetry of the fish. 



640 BACTERIAL DISEASES OF PLANTS 

ON INTERPRETATION OF PHENOMENA 

First of all, you are to remember that very often things 
are not what they seem! Two sets of phenomena may resemble 
each other superficially but be of quite unlike origin. Herein 
lies many a pitfall for the unwary. Probably most blunders 
in science result from failure to distinguish between similarity 
and identity, between resemblances that are fundamental and 
must depend on community of origin, and those that are only 
superficial and consequently must have diverse origins. The 
student, and the older worker as well, should be on his guard 
continuously against the fallacy of mistaken identity. The 
difference between a careful worker and one who is sometimes 
careless, or habitually so, lies in just this, that the latter sees the 
superficial similarity and is content with an inference, while the 
former probes the inference, demonstrates its non-validity, and 
saves his reputation. 

Laziness, or inhibitions due to overwork, lie at the bottom 
of most such blunders, I think, but sometimes over-confidence. 
Usually it is quite easy to show that a given result corresponds 
exactly to another or differs from it in various particulars, if 
environmental conditions are duplicated, and if cultures are 
made and sections are cut and studied, but all this takes time and 
painstaking care, which some persons are loth to give. It also 
involves good judgment and good training. Especially must 
you demonstrate, if you have made inoculations and obtained 
results: (1) that the resulting lesions are identical with those 
occurring naturalh' on the plant; and (2) that the organism 
in the lesions is identical with the one isolated from the natural 
disease and used for the inoculations. Not to do these two 
things thoroughly well is to leave your whole paper a tissue of 
uncertainties. 

"Verify everything!" is the best ad\dce I can offer. Then 
you will have no after regrets. Nearly every productive 
scientific man, however, has some regrets of this sort. 

ON REPETITION OF EXPERIMENTS OTHER PEOPLE's, ONE's OWN 

There is a mistaken notion abroad that if someone has 
worked on a subject and published a book or paper, that settles 



GENERAL OBSERVATIONS: ON REPETITION OF ENPERIMENTS 641 

it, and no one else need consider the problem farther, especially 
if that someone is a person of reputation. Xo supposition could 
be wider of the mark! Some reputations are founded on a rock, 
others are mere bubbles. ^Moreover, nature hides from us very 
securely her secret things, and the chances of going astray in 
their interpretation are many. The young scientific man, filled 
with his intellectual pride and knowing very little realh^ either 
about the complexities of nature or the histor^^ of science, which 
for the most part is the story of one long series of blunderings 
(toward the light, however, not into deeper darkness), is apt to 
judge the mistakes of his fellows and of older men harshl}"; the 
experienced honest man, on the contrary, Iftiows that to err is 
human and judges all honest work leniently, since he knows that 
even the best work is certain to contain some erroneous observa- 
tions, or some errors of interpretation. 

Remember this, therefore, as a fundamental doctrine in 
science: Nothing h too sacred to investigate, and nothing can be 
regarded as indubitably established until various careful observers 
and experimenters have arrived independently at the same conclu- 
sion. Copy this out and stick it up where you can see it every 
day! If the second man over a subject finds the first man correct 
in all essential observations and interpretations, the more credit 
to the former. The second man will, nevertheless, usually be 
able to extend the first man's observations somewhat and should 
leave the field clearer than he found it and in any event his ob- 
servations will be useful, as confirmation. Unfortunately, often, 
as the history of science shows, the second man over a subject is 
only a bumptious fool, and, when he has finished, the subject 
is covered with a cloud of uncertaintj-, until some third man, of 
greater ability, goes once more methodically over the entire 
field, blows away the dust, and again sets matters in their true 
light. If you repeat a man's experiments, try to be at least as 
painstaking and circumspect as he was, unless you wish to be 
intellectually pilloried for the contempt of oncoming genera- 
tions. Xever think it a waste of time or a work of superer- 
ogation to repeat the experiments of another person. Do not 
call it ''duplication of work." It is not that, because no two 
individuals ever bring to a problem just the same sort of train- 



642 BACTERIAL DISEASES OF PLANTS 

ing or outlook, and consequently, very often, one man finds what 
many others have missed. To exactly "duplicate" another 
man's work you would need to have exactly the other man's 
type of mind. Only by the labors of many minds has modern 
science come to stand where it does, as the only trustworthy 
interpretation of the world. 

So much, about the repetition of other people's experiments! 
It is still more important to repeat your own experiments. Most 
mistakes in science result from neglect of this simple and funda- 
mental precaution. If this practice were universal there would 
not be so many papers published, it is true, but those which did 
see the Ught would be much more worth reading, and would 
redound far more to the credit of the author and of the publisher. 
New species would not then be made from different shoots 
of the same root nor from different branches of the same tree. 
Remember: Rushing into print with some half -finished article 
may give you an ephemeral success, but not any lasting one! 
"Though the mills of God grind slowly, yet they grind exceed- 
ing small," and the clarified and final judgment of the world on 
any human performance is apt to be very near its true worth, 
certainly not in excess of it. Be careful then of what you 
publish. Repeat your experiments again and again, and only 
conclude that you have the truth when they advance each time 
to a definite result like clock-work. Then, righth% you may be 
full of that joy of discovery than which there are few keener 
delights, and may publish as speedily as possible with the full 
assurance that confirmation and due credit will not fail to appear. 

I now endeavor to repeat all my own experiments several 
times over and in the end I have a rounded-out and better view 
than one series only could possibly give me. Incidentally, I 
usually succeed in eliminating some errors or half-truths, which 
appertained to the first experiment. 

I consider this subject so important that the whole chapter 
ought really to be printed in capitals! 

Pasteur's two golden rules are worth remembering: N^a- 
vancez rien qui ne puisse etre prouve d'une fagon simple et decisive, 
and in the presence of failure, Refaisons les memes experiences, 
Vessentiel est de ne pas quitter le sujet. 



GENERAL OBSERVATIONS: ON PUBLICATION 643 

ON PUBLICATION 

The object of publication is to let other persons know what 
we have discovered. We cannot reach everyone, nor is that the 
aim, but we should be able to reach those cultivating the same 
field. The choice of a place for publication is, therefore, not 
unimportant. Generally we should choose some journal de- 
voted to our specialty or, at least, concerned with kindred topics, 
some publication in which one would naturally look for papers 
on plant pathology. Journals are better than Transactions 
because they are issued more regularh' and frequently, and are 
read more widely. Among journals select that recognized as 
a leading journal. If you print in a Transactions or in a Report 
be careful to select one that is published on time, not a year or 
two after going to press. Remember: printing is not publica- 
tion, but distribution by sale or otherwise is. By no means bury 
your contribution in a newspaper or other ephemeral sheet, 
nor publish it in a journal, or Transactions, that seldom prints 
pathological papers, lest it should be overlooked and perish 
still-born! Do not print it in the middle of some other man's 
paper, nor in the middle of one of your own papers devoted to 
some other subject. I am referring to actual cases! Print your 
paper, don't send it out mimeographed. Yet such copies are 
better than none. Finally, always secure and distribute several 
hundred separates, so that no one will have an excuse for neglect- 
ing it. In this distribution include all the leading journals and 
workers both at home and abroad. This is the more essential, 
in many cases, because certain journals have only a limited 
circulation outside of their own country, whereas science is in- 
ternational. Pathological problems also are international. I 
approve of patriotism, but that sort which has no international 
outlook is a narrow and vicious kind, fit only for barbarians. 

ON CLEARNESS IN PRESENTATION 

Having selected a place for publication, the serious question 
arises how best to present the subject matter. This is compara- 
tively easy only when the subject is a simple one and the contri- 



644 BACTERIAL DISEASES OF PLANTS 

bution is but a note. It is grave if the subject is complex, and 
the writing extensive. Moreover, I have observed that the 
difficulty increases in proportion to the ignorance of the writer. 
Many a big book could have been boiled down to a few chapters, 
and in some cases to a few sentences, or to nothing at all, had 
its author been possessed of clear ideas. As a means of com- 
pression, learn to think. This is too much to expect of every 
one, but not too much to insist upon for the man of science. 
Whatever is worth doing at all is worth doing well. Clarity 
is the soul of truth, and especially in science there should be an 
idea behind every expression, and this idea should be stated as 
clearly as language permits. To read the dictionary is usually 
considered in the light of a joke, but I doubt if any student could 
do better, and that, too, through a long series of years. If he 
does not continually thumb grammar and dictionary, and per- 
sistently read the best authors, he will seldom acquire a luminous 
and persuasive style, than which, exclusive of a single-minded 
devotion to the truth, nothing is more to be desired. There are 
various ways of saying things, but only one best way. Never- 
theless, to read the contributions of many scientific men one 
would suppose they must think any method of expression suffi- 
cient, even the most clumsy and ambiguous. Yet such is not 
the case. In spite of this motley array of bad writers, it is best 
that subject and predicate should agree, that one should avoid 
split infinitives and especially that each statement should be 
susceptible of but one interpretation! 

Every paragraph and every sentence in your paper should 
receive careful and repeated consideration, first, as to whether it 
tells the exact truth; second, as to whether it is absolutely clear, 
i.e., will convey the same meaning to all as to yourself (try it 
on your friends, if they will submit to it) ; third, as to whether 
it is complete, or requires various additions or qualifications — 
science is an eternal qualification; fourth, as to whether the 
sentences in it are entirely logical and move convincingly toward 
your final conclusions. These things can be determined only by 
repeated readings and much pondering. It helps greatly, when 
one has finished a paper, judging from my own experience, to 
turn back and re- write the whole of it. During this laborious 



GENERAL OBSERVATIONS: ON CLEARNESS IN PRESENTATION 645 

and more or less irksome process, many new ideas occur to me, 
and better ways of stating ideas already expressed. It helps 
also, I find, to put aside the completed paper and come back to 
it months later, as to a new subject, or to one by another author. 
Occasionally there is a person who can write a thing as it should 
be the first time trying, but I have known only one or two 
such persons. Generally, easy writing is hard reading. Dar- 
win sometimes recast his paragraphs a dozen times, and most of 
us may expect to reach a good style, if at all, only by dint of 
much labor and repeated re-writing. Yet who can doubt that 
it is an end worth all it may cost ? 

You publish to convince your readers and advance your 
own branch of science, and incidentally to enhance your own 
reputation. Look to it, then, that your writings are not only 
permeated with a love of the truth, but are forceful and limpid as 
a mountain stream. To this end, avoid technical terms when 
common words will serve, even if j^ou must do so at the expense 
of some conciseness. Nothing is more discouraging to the 
general reader than a book or paper bristling with a newly in- 
vented terminology, or full of mathematical formulae. 

ON COMPLETENESS OF PRESENTATION 

If you wait for absolute completeness, you will never publish 
anything but be always following up some one of the many side 
paths ramifying entrancingly in every direction from the great 
central subject under consideration. 

Nature is boundless and our own working lives are very 
short. There must, then, be some compromises. The investi- 
gation must be broken off somewhere. The question is, where? 
This is solved, partly, but not altogether satisfactorily, by not 
undertaking very complex problems. Ml I can say is — Do each 
piece of work as thoroughly as time permits, but publish, 
otherwise, especially on the assumption that you have something 
really worth publishing, your generation is more or less 
defrauded. 

Granted that you intend full publication, how complete the 
first paper should be, whether it should include all, or only 



646 BACTEKIAL DISEASES OF PLANTS 

a synopsis, or only some particular features of what is to follow, 
is a matter depending on various contingencies. If you have 
time, and are not likely to be forestalled, put it all into one com- 
plete and convincing paper, and illustrate it as thoroughly as 
possible. If, on the other hand, various other workers are in 
the field and you have reason to believe that their eyes are quite 
as sharp as your own, then it is important that you should get 
your discoveries into print as quickly as possible, if you are to 
receive due credit. You may then publish only a preliminary 
note, stating clearly what you have found and referring your 
readers to your later full paper for details and supporting proofs. 
Be sure of your facts, however, if you do this, since it is much 
better to let the credit of a discovery go to another than to rush 
into print only to discover later that you are wrong in places 
where you might have been right by taking a little more time 
for verification. In this connection it is well to recall the remark 
of the great zoologist of Johns Hopkins University, the late 
William Keith Brooks, when some one, alluding to an unpub- 
lished research of his, asked him if he did not fear anticipation. 
''I long since ceased to be troubled by such thoughts, for if 
another should publish on this or any other subject before I do, 
his work would probably be better or worse than mine. If 
it was better, I should be glad to be saved the mortification of 
having published poorer work; if worse, it would only afford 
additional material for my paper." 

This, I should say, is better advice for a mature worker 
with a well-established reputation than for a young man with 
his reputation to make, and yet it is worth the young man's 
pondering. 

By complete presentation I do not mean extensive and tedious 
presentation. Far from it! Many scientific papers, especially 
in Germany, are spun out to great length simply, it would seem, 
to increase the size of the honorarium. By all means avoid 
such doings. I shall deal more at length with this in the next 
section. 

ON BREVITY OF STATEMENT WHEN BREVITY IS NOT DESIRABLE 

A good rule is never to use two pages for a subject that 
can be compressed by a little thinking into one. The generality 



GENERAL OBSERVATIONS: ON BREVITY OF STATEMENT 647 

of men use more words to express an idea than are actually 
necessary, if the best words had been chosen. Study the mean- 
ing of words, their shades of meaning, and re- write a subject 
twenty times, if necessary, to state it cogently and with brevity. 
Remember: nearly everybody will read a brief statement on an 
interesting subject, while only the most phlegmatic and deter- 
mined will hold themselves to a long-winded one. You will 
more than treble the number of your readers by halving your 
paper! Moreover, for the necessity of those who can't spend 
even the minimum of time necessary to read a short paper, and 
for the convenience of everybody, especially of the foreigner, 
it is your solemn duty to sum up the substance of your contribu- 
tion in a series of brief conclusions which everyone will read, 
and which, if well put, may induce many to turn back and read 
your whole paper. No little thing vexes me more than to take 
up a paper two hundred pages long, let us say, often in a foreign 
language, and find no summary. I dip into it here and there 
trying to find what it is all about, without actually reading it 
word for word, and if I cannot do this the chances are that I 
throw it aside. Other people beside an author have some rights! 
Once I might have read it verbatim, but I have read too many 
such without profit, and now I am wary. It may be nearly all 
ambrosia, but how is one to know if its author has not respected 
it enough to provide a summary of its contents, as an appetizer? 
Study then with all your might how to be brief, how to say 
much in little, and do not use a word more than is requisite! 
Yet at the same time, use all the space that is necessary to follow 
your subject into all its various ramifications, and to present each 
and every feature of it clearly. Brevity is never desirable when 
it leads to obscurity. Often, especially in abstracts of papers 
read at scientific meetings, a few words more, especially if well 
chosen ones, would have converted a glittering generality which 
tells nothing, nothing exactly and usefully, and therefore is worth 
nothing, into a helpful note. There is a great opportunity 
for reform in this particular. Either journals should publish 
no abstracts whatever, or else exact, useful ones. Not every 
one can make a good abstract, in fact, very few can; and in 
general you should consult original papers rather than abstracts 



648 BACTERIAL DISEASES OF PLANTS 

if you would be well-served and master of your subject. Often 
it is some slight side remark of an author, sure to be missed by 
the reviewer, that will prove suggestive to you and fruitful. 

Another prevalent sin is neglect to provide long papers and 
books with a table of contents and with a suitable index. It 
is too much for any author to expect the reader to make an index 
to his book, unless he is a very guileless individual. My own 
opinion is that such authors are lazy, rather than unsophisti- 
cated. Any way, they deserve to be put into a pillory because 
sometimes unfortunately it is necessary to use their books, and 
to read much in order to get a little. Publishers are also to 
blame for accepting and printing unindexed works. That a 
second volume with a general index is contemplated is no proper 
excuse for neglecting to index the first volume, because the second 
volume may be long delayed or never published. I recall several 
such cases. Ebermayer's " Physiologische Chemie der Pflan- 
zen'' is a capital example. A second flagrant example is ''Les 
Maladies microbiennes des animaux" by Xocard and Leclain- 
che (2 Vols.. Paris, Masson et Cie., 1903;. 

ox THE ETHICS OF RESEARCH 

The scientific man is under the same moral obhgations as the 
rest of the world! He cannot plead "art for art's sake" and 
run amuck, but like the common man must be held to strict 
account. If he does disreputable things he must expect to 
suffer the consequences, and he mil, whether he expects it or not! 
The scientific man, of all men, ought to be the most upright, 
truthful and truthloving, because his whole life is spent in a 
search for the truth. If he cannot be trusted, who may be? 
If he has not high ideals, where shall we look for them? "Buy 
the truth and sell it not," should be his watchword. To him 
the truth should be a breastplate and a frontlet, a javelin and a 
strong tower. He should sacrifice to it and love and worship 
it above all things ! Therefore, when the scientific man departs 
from just ways the scandal is pecuharly great. For this reason 
and because the import of certain actions is not always realized 
at the time, especially by the younger workers, it is worth while 



GENERAL OBSERVATIONS : ON THE ETHICS OF RESEARCH 649 

to consider for a few moments the ethics of our conduct as re- 
lated to other scientific men, past, present and future. 

Credit to Earlier Workers. — From a single person, discovery 
seldom comes full-fledged, like ^linerva from the brain of Jove! 
Usually there are dim beginnings to which various men have 
contributed, and all of these, so far as they are real experimental 
contributions, should be cited by the man who is last to pubHsh, 
that the historic development may be plain. Indeed, it is im- 
perative that he should do so if he would avoid one or other of 
two inferences: (1) ignorance; (2) dishonesty. I will admit that 
to the uninitiated it makes an author seem more important, if 
no previous literature is cited in his book or bulletin or paper, 
since then he may be supposed to have done it all by himself, and 
this probably has been the incentive to some flagrant cases of 
omission, but a moment's reflection will show anyone that such 
a procedure is a very short-sighted one, particularly if the man 
desires the respect of his fellow-workers and of intelhgent laymen, 
who also soon learn the true state of the case. 

Begin your work, therefore, with a firm determination to be 
honest, and before you have gone very deep into any subject 
search out the literature of it and prepare a proper bibUography. 
Do tliis in a workmanlike manner, citing in full — author, title, 
place of publication, year, volume and page, and make it chrono- 
logical not alphabetical. A chronological bibliography shows 
the development of a subject at a glance and this is what the 
student desires to know, or should desire to know, since this is 
the historical method. Yoiu" readers will thank you earnestly 
for full citation, and. on the other hand, will curse you. if you 
carelessly refer them to places where the article is not to be found, 
or where it can be found only after prolonged search, generally 
in time that can be ill-spared for it. ^lany a day have I wasted 
tr\dng to run down slovenly bibhographical references, and hence 
r write with some feeling. Every author owes certain things 
to his readers and this is one of them. It is so easy for you to 
fix a citation right when you have the volume and page before 
your eyes and so difficult for another to verify it when he has 
only some bimghng assininity as his guide. 

You must know the hterature of your subject. whate\er else 



650 BACTERIAL DISEASES OF PLANTS 

you neglect and must cite it, and as a rule you should see, as I 
have said, the original papers. To this end, learn several foreign 
languages and have accurate translations made of such impor- 
tant papers as you cannot read in the original. 

Finally, you must not minimize the work of the earlier author 
to magnify your own work. This is a common and mean sin. 
Such meanness of soul is its own reward, but always there is 
further punishment in store, since by no shift can such a person 
avoid the moral judgments of his own generation and of pos- 
terity. When a man has made use of an earlier author without 
citation it would seem that the work is his own, but often he has 
copied not only the discoveries but also the peculiar mistakes 
of the first writer and thus his dishonesty is revealed and 
punished. 

Treatment of Contemporaries. — There is much honest 
rivalry in science and to this there can be no objection what- 
ever. No one has any right to build a tight board fence around 
any locality or any problem and claim it as his peculiar pre- 
serves. Such a claim is highly absurd. Nature is free, or should 
be, for all, and the more outspoken the criticism and frequent 
the rivalry, the more rapidly will science advance. Nothing 
throws such a wet blanket over the advancement of science as 
the suppression of free speech and the domination of a few would- 
be masters. Feel perfectly free, therefore, to take up any prob- 
lem that interests you, unless you know that some one else has it 
already well in hand, in which case courtesy would seem to dic- 
tate the choice of another subject. The mere fact, however, that 
others have begun to work on it need not deter you, if you are 
not beholden to them for any of your ideas, and know that their 
researches are as yet only in the dough, and this is peculiarly 
true if the disease is wide-spread and economically important. 

The things which you must not do are these: You have no 
right to begin a research, or to finish one, building it on the 
unpublished ideas of another man which he has imparted to you 
in confidence, or which you have obtained in some illicit way. 
It may be you have overheard a conversation, or have seen by 
accident some of the other man's experiments, or have received 
information from a friend who has heard the news or seen the 



GENERAL OBSERVATIONS: ON THE ETHICS OF RESEARCH 651 

experiments. By use of this knowledge you may be able to 
fructify your own sterile ideas and forestall him, but you have 
no moral right to do so. You cannot use these ideas without 
low^ering j^ourself in your own estimation and in that of your 
fellows, for stealing, like murder, will out! You cannot conceal 
it, try as you may! 

Be something first, then do something! Be willing to stand 
upright and to build on your own foundations. 

Don't listen to conversations not meant for you, don't go 
about poking and prying, don't ask leading questions, and if 
anyone seems about to volunteer information as to his own work, 
ask him not to tell you. Thereby you may avoid charges of sup- 
planting another, and much bitterness, because it often happens 
that other workers tell you the very things you already know, 
and have known perhaps for years, and yet if you then publish 
them as your own, the other man will be pretty certain to think 
that you are indebted to him and that you have not dealt 
wdth him justly. Judging from my own case, every experi- 
enced worker has hundreds of unpublished facts not of much 
value by themselves, perhaps, but kept in store waiting the dis- 
covery of other facts necessary to weld them into a vital w^hole. 
I give opinions freely where I can, and spend an aggregate of 
much time in the service of others, but to every one who comes 
to me for advice and wishes to tell me just what he has done, I 
now say, if he is working in my own field: "Don't tell me! 
Keep it to yourself until you have published it; then I shall be 
glad to read it." 

You also owe to your fellow worker certain minor amenities. 
One of these is courtesy, another is generosity. If you discover 
scientific material of value to him, and which you do not in- 
tend to use, it is your duty to send it to him. Also, if you run 
across out-of-the-way papers on his subjects, he will be grateful 
to you for a reference and the kindness costs you but little. 
Finally, do not bother him with unnecessary questions, or ask 
for his unpublished data, or for his cultures until he has published 
on them, or for information it may take him days to prepare for 
you. Young men are often very inconsiderate in such matters, 
particularly if they are also very ignorant. I have had a re- 



652 BACTERIAL DISEASES OF PLANTS 

quest within the year for references to all of the literature of 
plant pathology. Such requests, coming now and then, elicit 
only a smile, but dozens of them get to be a bore. 

What We Owe to the Future. — Our duty to posterity re- 
quires us to pass on the torch of learning, trimmed and burning 
brightly. The little we are able to do individually brightens or 
obscures the pathway of science just to the extent that we are 
honest or dishonest, penetrating or dull. Our duty to the future 
is to do things thoroughly, to state things clearly, to point out 
weak places in our own work as freely as in that of others ; and to 
cover up and obscure defective spots, for our own immediate profit 
or reputation, under no circumstances whatever. If you have 
made a mistake, the manly way is to own it. Shame should 
not deter you, for every one makes mistakes, even the most 
famous and the most circumspect : Darwin made them, Pasteur 
made them. The only man who never makes any mistakes is 
the one who never makes any discoveries. 

Ideals. — You are to remember that you are consecrated to 
the truth. If that prevails and you have helped to make it 
prevail, what matters it if you are first in the race, or last? 
In Milton's noble words, ''They also serve who only stand and 
wait!" Nevertheless, the workman is worthy of his hire, and 
the great public is too little cognizant of this fact when that 
workman is a scientific man and not the director of a corpora- 
tion. Science and the scientific man have not yet come to their 
own. The life of the common man in modern times has been 
ameliorated in a thousand ways by the labors of scientific men 
but he seldom thinks of this, and the Croesus never! It is a 
part of our duty to demand justice and fair play and to organize 
to get it if necessary. 

Cultivate good judgment, be not easily discouraged, confess 
ignorance, aim high, be diligent! What Montaigne said about 
learning in general, applies with peculiar force to science — La 
science, pour bien faire, il 7ie faut pas seulement la loger chez soi, 
il la faut epouser. In his fascinating chapter "On books," 
Montaigne also has the following words of wisdom which are 
well worth taking to heart: La scie7ice et la verite peuvent loger 
chez nous sans iugement; et le iugement y peult aussi estre sans 



GENERAL OBSERVATIONS: IDEALS 653 

elles: voire la recognoissance de Vignorance est Vun des plus beaux 
et plus seurs tesmoignages de iugement que ie treuve. 

Perhaps I can best close this section with the mottoes of 
my own laboratory which are from far away and long ago, but 
which I love all the more for these reasons, since in my phi- 
losophy of life all men are brothers and all times helpful. We can 
never escape from the great Past if we would, and we ought not 
to wish to do so, because to it and to the great men of other 
races than our own we owe many things — reverence being one. 

"We are the fruit of Time and owe all to the imnieasurable Past." Emerson. 

I love them also because they teach at the same time up- 
rightness and humilit}', independence and co-operation. The 
first is from Egypt and the second from China: 

Be not of your learning vain. 
Treat the simple and the wise 
With like honor. Open lies 
Art's great gate for all, and they 
Who have entered by that way 
Know how still before them flies 
The perfection they would gain. 

Precepts of Ptnh-Hotep. 
(33 centuries B.C.) 
Sayings of Confucius 
(500 B.C.) 

The way of heaven and earth may be completely disclosed in one sentence — 
they are without any doubleness. 

To be fond of learning is to be near to knowledge. 

The essence of knowledge is, having it, to apply it; not having it. to confess 
j'our ignorance. 

There were four things the Master taught: Letters, ethics, devotion of soul 
and truthfulness. 
The Doctrine of the Mean. The Confucian Analects 

There are many equally wise and suggestive aphorisms in 
the Confucian books. Here is one: 

Hwuy gives me no assistance. There is nothing that I say in which he does 
not delight. 

And here is another: 

Yuen Jang was squatting on his heels, and so waited the approach of the 
Master, who said to him, "In youth not humble as befits a junior; in manhood 



654 BACTEKIAL DISEASES OF PLANTS 

doing nothing worthy ot being handed down ; and Uving on to old age : This is to 
be a pest." With this he hit him on the shank with his staff. 

Look for the "1j. Y. T." edition, English-Chinese, published 
at Hongkong in. 1898. Buy it, if you can find it, and live 
with it. 

ON KEEPING one's OWN COUNSEL 

The scientific man's discoveries are his stock in trade. On 
them depends his reputation, and on that, very often depends 
his ability to obtain a livelihood and to care for those dependent 
upon him. The pecuniary compensation he receives is small, 
often despicable, if we consider the initial ability required and 
the long years of training requisite to perfect it into a productive 
career, and, especially, if we compare it with the great and allur- 
ing rewards held out to the young man by professional and busi- 
ness opportunity. Among a multitude of dollar chasers, who 
usually despise him, the scientific man deliberately chooses to 
remain poor for love of his science. His discoveries are his sole 
riches! He has no regrets and does not ask for sympathy but 
would like justice, and a livelihood! Any course of action, 
therefore, either on his own part, or on the part of directors or 
boards of control of institutions, which tends to rob him of the 
results of his labor strikes him in a peculiarly vital manner. 
If his discoveries are stolen or are frittered away to others 
by premature publicity, he is robbed of all that which makes 
his professional life worth while. Money he has not, and 
reputation is taken away from him. There is very little appre- 
ciation of this fact on the part of non-scientific men, but it 
is a fact, nevertheless, and one which must be taken into account 
by all who have to deal with men of science. Their ideals and 
their psychology must be understood and should receive courte- 
ous consideration from all in authority over them if they are to 
work without endless friction and deep humiliation. Most of all, 
the safety of his unpublished researches must be considered 
by the scientific man himself since he is beset on all sides, first, 
by marauding fellow workers who being unable to get results 
themselves are always on the lookout for what crumbs they may 
be able to pick and steal from others; second, by the so-called 



GENERAL OBSERVATIONS: ON KEEPING ONE's OWN COUNSEL 655 

practical man who is generally clamoring to have his own selfish 
needs satisfied without much consideration for the research 
worker. He thinks it enough if the scientific man's salarj^ is 
paid quarterlj^, yet there are more important obligations than 
payment of salary. The number of scientific men who deliber- 
ately steal from their fellows is few, I should hope, yet a half 
dozen glaring instances of this sort of iniquity have come to my 
knowledge in recent years and lead me to believe that his dis- 
reputable kind must be always wandering about. The scientific 
man must, therefore, protect himself as best he can not only from 
this class but from that much larger class who have some ideas of 
their own, which, however, require the stimulus of another's 
speech to render them fertile, and who seldom trace their in- 
centive to its real source, or give any thought to it. We are 
all more or less of this sort, and while protecting our own discover- 
ies should also avoid receiving what properly belongs to another. 
These remarks lead naturally to the following piece of 
advice, viz., when j^ou are with men working independently in the 
same field or with their friends — keep your own counsel. You 
may talk freely with them on apparatus, technic, the last 
novel, or any subject on which you are not ivorking, but never a 
word on what you have discovered, or are now doing! It is time 
enough for them to know when you publish to the world what 
you have discovered. Conversely, a dehcate sense of propriety 
should lead you to avoid trying to discover what the other man 
has done, or is doing. In other words : Don't talk shop with 
visitors. Any other course is suicidal. Nor should visitors be 
allowed to wander freely about experimental houses, laboratories, 
or grounds since an inoculated plant or animal may speak plainer 
than a man on a house-top. Cases are known where even the 
reading of a paper in advance of its publication has given another 
man opportunity to lay fraudulent claim to priority of discovery, 
with resulting loss, recrimination and bitterness. For this 
reason papers detaihng important discoveries should not be read 
before societies until they are actually in type and ready to 
appear. It is not a good plan even to tell what you contem- 
plate doing. Do it, rather than expatiate on it, and when it is 
done then quickly make it available to every one. 



656 BACTERIAL DISEASES OF PLANTS 

Pasteur set an admirable example in this respect, as in 
many others. He had no confidants. No one outside of his own 
laboratory, and often not even his assistants, knew what he 
really thought about a problem until the time was ripe for the 
announcement of his perfected discovery. No one suffers from 
such a course because discoveries published in an incomplete 
form, mixed with various erroneous assumptions incident to the 
early stages of a research, are seldom very useful. It is better to 
hold back the report until everything is verified and then so to 
publish it that the man who has actually made the discoveries 
shall receive the credit which he deserves, and by it be stimulated 
to make additional discoveries. It is all the more striking and 
memorable if these discoveries come like a flash out of a clear sky ! 

This brings us to the obverse side of the shield, which 
will be considered in the next section. 

ON TEAM WORK 

Nothing in the foregoing chapter militates in any way 
against the association of scientific men and women in small 
groups for the easier solution of difficult problems. Many com- 
plex problems can be attacked successfully only in this coopera- 
tive way, and this has come to be very clearly understood in 
modern times and is now, I think, rather overdone, there being a 
disposition in some quarters to consider scientific men as only 
so many cogs in a mechanism to be run exclusively by a non- 
scientific director or business man, in the interests of a blear- 
eyed and jealous god called ''Efficiency." The highest eflSciency, 
however, obtains when the non-scientific man does the least 
meddling. ''The accomplished scholar is not an utensil." 

Such team-problems involve mathematics, chemistry, physical 
chemistry, physics, and various phases of biology and no one 
person can be supposed to possess the requisite training in all 
of these sciences, but several congenial persons by joining forces 
may succeed unitedly where each would fail separately. I have 
used the word "congenial" intentionally, for inharmonious 
natures do not mix any better than oil and water. Not every 
person is adapted to team work. Indeed, some persons are so 
suspicious of every one that they are shut into a very little world 



GENERAL OBSERVATIONS: ON TEAM WORK 657 

of their own and seldom accomplish much. These are at just 
the other extreme from those expansive persons whose loquacity 
is their worst enemy. There is, however, a happy middle ground 
where justice and mutual esteem prevail. 

Usually in team work there must be a leader, if the various 
separate researches are to be properly coordinated, and always 
there must be frequent conferences and a mutual good under- 
standing. This generally involves proximity. It is of little use, 
ordinarily, in my judgment, to undertake long-distance coopera- 
tions in biological research. Either money is wasted by lack 
of careful planning and constant supervision, or one party gets 
the lion's share of credit by publishing in advance of and without 
the consent of the other party or, finally, one does most of the 
work or makes most of the discoveries and yet must share 
equally with the shirker, or with the dull one. Such coopera- 
tions are always a loss to one of the contracting parties, and 
frequently also, from neglect on the part of one of the coopera- 
tors, a loss to science. Said a well known experiment station 
director to me some years ago: "If I have discovered any good 
thing I am going to keep it to myself; if you have discovered 
any good thing, and wish to share it, I shall be very glad to 
'cooperate.'" I also heard another station director say in 
public, frankly and without shame, that he wished to get all of 
the Government money he could for expenditure in his own State, 
but that he cared nothing for researches outside of it. This, 
I believe, is the kind of cooperation many people have in mind, 
but it is not the sort that makes for the advancement of science, 
or that which I have in mind. In true team work each party 
does his share honestly and efficiently and the credit belongs 
equally to all. Nevertheless, the most brilliant and far-reach- 
ing discoveries are usually the product of a single mind. 

The field applications of research, on the contrary, afford 
many opportunities for useful cooperations in plant pathology. 

ON SHARING CREDITS 

This is a delicate subject and views differ, especially the 
views of younger and older workers. What I shall have to say 
belongs logically under two heads. 



658 BACTERIAL DISEASES OF PLANTS 

Teachers vs. Pupils. — A thousand interesting problems occur 
to every live teacher, but, with his hands tied by a multiplicity 
of duties connected with the imparting of knowledge, he must 
carry on his own researches in the odds and ends of his time, and 
largely by setting various students at work on these problems. 
They are so many instruments of inquiry, rather dull and defec- 
tive tools in many cases, it must be admitted, but better than 
none. The teacher cannot be blamed, therefore, if he uses his 
students in this way. They are but beginners. They cannot 
do researches alone, and even with much guidance they generally 
manage to plunge into every alluring pitfall, every bog that in 
the least resembles solid ground. If the teacher is competent 
and faithful they receive from him far more in the way of stimu- 
lus and training than the value of what they return to him either 
in money or in research. Each student discovers something, 
let us say, but only the labors of many students in various 
years, plus the insight and the additional labors of the professor 
enable him finally to present a finished piece of work. Mani- 
festly, the completed work belongs to the teacher who has been 
the brains of it from the beginning. Certainly it does not belong 
to the individual students, many of whom, perhaps, are now 
specializing in quite other fields or have abandoned science 
altogether, having gotten from it the training desired. In excep- 
tional cases where the student has developed marked aptitude 
for a research, has devoted an unusual amount of time to it, 
and has made independent discoveries, the teacher should, I 
think, share with the student in the finished product, and gen- 
erally I beheve he is willing to do so. Students are often rather 
conceited and not always just to faithful teachers. It is good lo 
try to put one's self mentally into the teacher's place. It is 
also good to remember St. Paul's advice — "Let no man think 
more highly of himself than he ought!" Modesty is commend- 
able in all, and especially in intellectual babes and sucklings. 
These remarks apply in large measure also to graduate work 
exclusive perhaps of "doctor's theses," of which in my judg- 
ment there are altogether too many published. 

Chiefs vs. Subordinates. — In early stages of association 
the case is the same between chiefs and assistants. Later when 



GENERAL OBSERVATIONS.* ON SHARING CREDITS 659 

the apprenticeship has been fully served, proper training ac- 
quired, and ability for independent research developed, there 
should be a just sharing of credits. Any other course leads to 
injustice and bitterness, is not creditable to the chief, and should 
not be endured by the subordinates. Here again, however, the 
subject calls for an exhibition of mutual forbearance and 
courtesy. In the end, the aim of all wise and honest chiefs 
should be to see their assistants set up as independent workers — 
honest, capable and productive. It is enough glory for him that 
he has trained them! All this on the supposition that they have 
developed marked ability for independent research. The time 
required for this development varies greatly, and some never 
acquire it. The latter must be content to serve always in sub- 
ordinate places, and all should defer a good deal to the judg- 
ment of their chief, premising always that he is an honest man 
of broad views and sound judgment. 

ON ATTENDING MEETINGS AND KEEPING UP MEMBERSHIP IN 

SOCIETIES, AND ON BEING GENERALLY PUBLIC-SPIRITED 

AND HELPFUL IN SCIENCE 

A man's success in life, granted some inborn ability and 
a proper training, depends very largely on the friends he makes, 
both the number and kind. If he is a reserved and shy individual, 
he is apt to get on slowly. He may be an excellent man but 
nobody finds out his good qualities. Likewise, if he is penurious, 
he stands very much in the way of his own advancement because 
then naturally he will think he cannot afford to purchase and 
distribute separates of his own papers, to buy books and to sub- 
scribe for scientific journals through which he would become well 
informed, or to belong to societies and attend meetings where he 
would meet many interesting men and might make friendships of 
lasting service to him. A clear outlook is essential. Remember 
the proverb: '' Nothing ventured nothing won! " If you plan a 
career in science you cannot do better than to join scientific 
societies and attend scientific meetings regularly, even at cost 
of considerable inconvenience and self-denial. You should also 
visit other laboratories, and strive in every way to keep abreast 



660 BACTERIAL DISEASES OF PLANTS 

of the rapidly moving current of modern scientific life. It is a 
duty that you owe not only to yourself, but also to your fellow- 
workers, since no one lives wholly to himself! Science has 
many enemies, and being your chosen goddess deserves your 
unqualified and generous support. Her worshippers are a small 
band and often they are too individualistic for their own best 
interests. In union only is there strength. If you are not 
public-spirited and helpful, you fall naturally into the ranks of 
the mean and selfish, and will deserve the fate you may be quite 
sure the gods have in store for you. It may even now be upon 
you without your knowing it. 

On the contrary, if you are public-spirited, friendly and 
generous, lending a hand wherever you can, working along 
patiently and thoroughly, biding your time and not disturbed 
by selfish elbowing or band-wagon tactics on the part of 
your fellows, your opportunity will surely come, and a hundred 
hands will be reached out to help you where you expected none 
at all. The scientific public is quick to welcome all worthy 
comers, only it must be certain that they are really worthy, and 
if you keep yourself away from your fellows, how can anyone 
know what stuff is in you? 

Be, then, ambitious for a worthy place and be willing to 
work hard for it, sixteen hours a day if need be, in spurts, but 
also remember that you must let people know who you are and 
what you are doing. 

The counter of this advice is — Do not crowd in where you 
are not invited, do not elbow and push for the best places, but 
rather strive to be so courteous and to make yourself so thor->' 
oughly master of your subject that people will invite you. ^^ 

Honors in science are welcome if they come unsought, but, 
always, if you lobby for them, they will be less pleasant than you 
anticipated, and will lower you in your own estimation and in 
that of others. 

ON REST AND RECREATION 

By good food, pure air and temperate habits seek to put 
your body into tone, like some perfect musical instrument. 
Then the joy of living will overflow, expressing itself in ener- 



GENERAL OBSERVATIONS: ON REST AND RECREATION 661 

getic good work. Juvenal's mens sana in corpore sano is the 
ideal! Especially be very careful of your eyes since they are 
your most precious instrument of research and avenue of infor- 
mation. Very often, unknown to the student and the man of 
science, bad headaches result from slight eye-strain. For this 
reason consult good oculists frequently and, in general, avoid 
reading in bed, on a moving train, in fading daylight, or by a 
dim or flickering artificial light. Use your eyes interchangeably 
at the microscope and rest them frequently if the instrument 
tires. Properly used, the microscope ought to strengthen the 
eyesight, or at least ought not to harm it. 

Finally, remember that much use dulls a delicate instrument, 
especially the brain, while frequent rest and recreation tone up 
the mind to keener insight. Years ago a well-known man of 
science told me that two of his most interesting discoveries were 
made on days when he had decided to do nothing and had gone 
out to lie under the trees. The late Sir WiUiam Osier said to me : 
"I work tremendously hard nine months in the year, but the 
other three I play." Change of scene often means renewed life 
and energy and even change of subject helps somewhat. Many 
scientific men are rather narrow in their outlook on life and would 
be greatly improved not only socially but in every other way, 
by getting at frequent intervals entirely away from their narrowing 
specialty, through the cultivation of hterature, music, art, nature 
in her more general aspects, and the amenities of social life. 



/ 



INDEX 



Acetic acid tumors, 483, 555 

figures illustrating, 488, 490, 491 

492, 493, 494, 495, 555 
hypertrophy only in early stages, 

of, 555 
origin of cells of, 555 
substomatal injury preceding, 
503 
Acids, found in fruits, table of, 13 
increase of, in tumor-forming tissues, 
531, 543 
Adventitious buds, 50, 419, 574-630 
Agar used for culture media, 105 
Air in tissues, how removed, 295 
Alcoholic material, care of, 120 
Aldehyd, tumors formed on plants by, 
483 

figures illustrating, 497 
Alfalfa 

bacterial disease of, 55, 474 
white spot (physiological disease), 55 
Amenities of research, 651 
Ammonia, tumors formed on plants by, 
483, 553 
figures illustrating, 486, 487 
loss of water precedes forma- 
tion of, 489 
theoretical action of, 567 
Angular leaf-spot 

of cotton. (See under Cotton) 

55, 314 
of cucumber. (See Bacterium 
lachrymans), 
Animal and human pathology, useful 

to plant pathologists, 635 
Ants, control of, 109, 114 
Aplanobacter, morphology of, 35, 132 
Aplanobacter agropyri O'Gara, 47, 473 
Aplanobacter michiganense EFS, 202 
behavior on various media, 206 
cause of bacterial canker of tomato, 
202 



Aplanobacter michiganense EFS, char- 
acters of, 205 
geographical distribution of, 205 
in both field and hothouse, 202 
infectious nature of, 219 
isolation difficult, 206, 207 
literature, 222 

meristematic tissues attacked, 207 
method of obtaining pure cultures, 

207 
phloem infected by, 202, 208, 209 
resemblance on cooked potato to 

Bacterium campestre, 206 
seed-infection, 202, 216 
sensitiveness to acids, 211, 217 
stomatal infections, 202 
Aplanobacter rathayi EFS, 473 

distortions in infected plants, 49 
resistance to sunlight and drying, 

36 
Aplanobacter sepedonicum (Spk.) EFS, 

207, 474 
Aplanobacter Stewarti (EFS) McC, 

(See also Stewart's disease of 

maize.) 
characteristics of, 160 
colonies of, 172, 173 
color changes in infected corn 

plants, 49, 162 
cultural characteristics, 161 
description of, 161 
host plants, 167 
inoculation methods, 165 
literature, 176 
morphology of, 161 
most susceptible age of host, 8 
resistance to drying, 36 
seed-borne, 161, 174, 176 
technic for obtaining pure cultures 

of, 163 
temperature relations, 161 
varieties of maize sensitive to, 167 



663 



664 



INDEX 



Aplanobacter stewarti, virulence per- 
sistent, 161 
Aplanobacter teutlium (Metcalf) EFS, 

474 
Apparatus for 

hothouse and inoculation experi- 
ments, 86 

isolation and care of cultures, 80 

photographic room, 89 

preparation and study of sections, 81 

preparation of culture media, 77 
Appel's potato-rot (See Bacillus phy- 
tophthorus) 
discovered in Germany, 64 
Apple, fire-blight of (See Bacillus 

amylovorus) (See also under Fire- 
blight), 359 
Ardisia, experiments with, 44, 45 

figures illustrating, 41, 42 

mutualism in, 41, 42 
Arthur (J. C), 2 
Ascobacterium luteum Babes, 344, 395, 

411 
Ash tumor, 391 

Australia, bacterial diseases found in, 52 
Autoclaves, for sterilizing soil and pots, 
86 

in use in pathological laboratory, 78 

Bacillus amylovorus (Burrill) Trevi- 
san. (Cause of fire-blight of 
apple, pear, quince, etc.), 359 
acids from sugars, 371 
anaerobic (facultative), 369 
appearance of infected trees, 359, 

360 
blossom-infection, 360, 374 
cankers on pear and apple trees 

caused by, 360 
colonies of, 378, 379, 381 
control measures, 377, 385 
description of, 367, 374 
economic importance of disease 

due to, 365 
erroneous statements respecting, 

373 
factors favoring spread of, 13, 360, 

365 
flagella, 367, 377 



Bacillus amylovorus, germicides, 71, 
365, 386 
growth on or in, 

agar plates, 370 
beef bouillon, 370 
Cohn's solution, 371 
gelatin plates, 370 
milk, 370, 382 
potato broth, 370 
Uschinsky's solution, 371 
hold-over blight due to, 360 
host plants, 359 
in bark, 359, 365 
inoculation experiments, 374 
in sap wood, 381 
liquefying, 369 
literature, 387 
losses in United States due to, 54, 

55 
method of isolating pure cultures 

of, 374 
methods of control, 71, 365, 377, 

385 
most susceptible age of host, 8 
non-gas forming, 370 
non-nitrate reducing, 367 
ooze from hold-over blight, 360, 367. 

368 
ooze from summer blight, 369, 370, 

383 
optimum temperature for growth, 

371 
plants susceptible to, 359 
pruning to check, 367, 376 
resistant varieties, 377, 385 
ripe tissues immune, 372 
secondary infections of, 363 
susceptible varieties, 377 
thermal death point, 371 
transmission by insects, 360, 384 
use of cyanide for disinfection, 

71 
use of formalin for disinfection, 71 
use of mercuric chlorid for disin- 
fection, 365, 386 
virulence persists, 365 
winters over in trees, 360 
yellow saprophytes may accom- 
pany, 374 



INDEX 



665 



Bacillus apiovorus Wormald, 67, 239, 

241, 244, 245, 246, 474 
Bacillus aroideae Townsend, 240, 247, 
248 
behavior in milk, 250 
Bacillus atrosepticus Van Hall, 265, 266 
Bacillus carotovorus L. R. Jones, (See 
also soft rot of carrot), 223 
acids produced by, 230, 238 
attacks young green shoots of car- 
rot, 237 
behavior in various media, 230, 237 
behavior in the tissues, 223, 224 
cause of soft rot of carrot, 223 
colonies of, 

on agar, 232, 236 
on gelatin, 239, 240, 269, 276 
color on media, 230 
compared with other soft rot 

organisms, 239, 240 
disintegration of cell-wall, 232 
dwarfing of plants infected with, 

49, 228 
flagella of, 234 
gas formed, 230, 237, 240 
gelatin liquefied, 234 
grape sugar, harmful to, 251 
host plants, 223, 225, 226, 229 
inoculation experiments, 241 
involution forms, 238 
literature, 252 
means of gaining entrance to the 

plant, 223 
method of isolating from the tis- 
sues, 241 
milk curdled, 234 
milk-rice, 246 
nitrates reduced, 230 
range of cultural characters in 

doubt, 232-241 
resemblance to other organisms, 

223, 229, 240 
sensitiveness to drying, 36, 147, 231 
sensitiveness to sunlight, 36, 231 
swelling of cell-wall, 251 
technical description, 230 
temperature relations, 231, 238 
tolerates sodium chlorid, 238 
type of disease caused by, 223 
virulence not lost readily, 229 



Bacillus carriers, 33 

Bacillus coli Esch., 54, 355, 356, 408, 474 

Bacillus cubonianus Macch., 342, 344, 

346 
Bacillus delphini EFS, 474 
Bacillus gossypina Stedman, 314 
Bacillus harai Hori and Miyake, 53, 474 
Bacillus mangifera Doidge, 473 

figures illustrating, 3, 9 

most susceptible age of host for, 8 
Bacillus melanogenes Pethybr. and 

Murphy, 265, 268 
Bacillus melonis Giddings, 240 
Bacilhis musae Rorer, 473 

figure illustrating effect of, 11 

type of infection. 10 
Bacillus oleae (Arch.) Trev., 394, 396 
Bacillus oleae-tuberculosis Savastano, 

394 
Bacillus oleraceae Harrison, 239 
Bacillus omnivorus Van Hall, 239 
Bacillus phytophthorus Appel (See also 
Bacterial black rot of potato), 
253 

acid and gas forming, 257, 260, 263 

alcohol, effect of, 260 

appearance of tissues infected with, 
255 

behavior in various media. 257, 260 

cause of potato black rot, 257 

colonies of, 269, 270, 271, 272 

distortion of infected potato plants, 
49 

dry air sensitive, 257, 278 

flagella of, 267 

gelatin liquefied, 257 

inoculation experiments, 267 

inosite, gas from, 263 

lactose, gas from, 263 

life in soil, 34 

literature, 278 

mannit, gas from, 263 

method of isolating pure culture of, 
263, 266 

milk curdling, 257 

milk-rice, 246, 260 

nitrate reducing, 257 

non-growth in Cohn's solution, 260 

non-parasitic coccus accompany- 
ing, 263 



666 



INDEX 



Bacillus phytophthorus Appel, raw 
potato, growth on, 264, 267 

ready means of identification, 267 

related organisms, 265, 268 

technical description, 257, 260, 263 

type of disease, 253 

virulence of, long continued, 34, 35, 
258, 263, 275 
Bacillus solanisaprus Harrison, 265, 268 
Bacillus spongiosus Aderh. and Ruhl., 

474 
Bacillus tracheiphilus EFS. (See also 
Cucurbit wilt), 132 

anaerobic (facultative), 135 

cause of cucurbit-wilt. 132 

coccus follower of, 138 

colony, internal markings of, 136 

colony, surface appearance of, 138 

cultural characters, 135 

dwarfing of plants infected with, 49 

figures illustrating, 136, 138, 140, 
143, 144 

flagella of, 136 

geographical distribution, 132 

host plants, 137 

insect carriers of, 30, 134, 135, 144 

isolation of, 135 

literature, 145 

sensitiveness to acids, 135 

sensitiveness to drying, 36, 135, 147 

sensitiveness to sunlight, 36, 135 

staining of, 117 

technic of inoculation, 137 

type of disease, 132 

viscidity, 136 
Bacteria in plants 

action on middle lamella of cell- 
walls, 46 

animals inoculations with, 36, 459 

cultural characters of, 35, 36 

gas production of, positive, 36, 230, 
257 

gas production of, negative, 135, 
147, 161, 182, 205, 306, 347, 369, 
396, 421 

intracellular, 46 

many diseases due to, 1, 4 

morphology of, 35 

most, extra-cellular, 46 

pigments of, 35 



Bacteria, production of toxins by, 46 
small size of, 75 
staining of, from cultures, 118 
staining of in tissues, 116 
Bacterial black rot of potato, 253 
Bacterial canker of tomato. (See 
Aplanobacter michiganense), 
202 
a phloem disease, 202, 209 
figures illustrating, 203, 204, 205, 

206, 210, 211, 212 
histology, figures illustrating, 

207, 208, 209, 213, 214, 215 
Bacterial cell, why able to do so much 

work, 515 
Bacterial diseases of plants 

changes of susceptibility to, 9 

cryptogams, occurrence in, 4 

discover}' of, 1 

distortion of host plant as a re- 
sult of, 49 

distribution of, among flowering 
plants, 4 

dwarfing of host as a result of, 49 

early workers on, 1 

fungous diseases following, 34 

geographic distribution of, 51 

immature tissues and, 8 

in Australia, 52 

in China, 53 

in Denmark, 62 

in Dutch East Indies, 52 

in France, 66 

in Germany, 64 

in Great Britain, 64 

in Holland, 62 

in India, 54 

in Italy, 66 

in Japan, 53 

in New Zealand, 55 

in the Philippines, 54 

in Portugal, 66 

in Russia, 66 

in Sandwich Islands, 64 

in South Africa, 54, 55 

in South America, 54 

in Spain, 66 

in Tasmania, 55 J 

in United States and Canada,_54 

in West Indies, 52 



INDEX 



667 



Bacterial diseases of plants, incuba- 
tion period of, 17 
latency of, 8, 17 
list of plant families attacked by, 

4 
list of plant genera attacked by, 

7 
losses due to, 51, 52, 53, 54, 61, 

62, 64, 66 
matured tissues attacked by, 9 
methods of control, 68 
new type of, 48 
number of, 1, 4 
one followed by another, 34 
parenclwmatic vs. vascular, 10 
period of greatest susceptibility 

to, 8 
reaction of host plant to, 48-51 
scepticism as to occurrence of, 1 
slow early progress in study of, 1 
subject forty years old, 1 
what governs infection, 12 
when first discovered, 1 
Bacterium, morphology, 132 
Bacterium andropogoni EFS, 473, 475 

method of infection, 16 
Bacterium aptatum Brown and Jamie- 
son, 474 
green fluorescent species, 36 
Bacterium atrofaciens McCuUoch, 474 
cause of basal glume rot of wheat, 

55 
figures illustrating, 57, 58 
Bacterium beticolum Smith, Brown and 
Townsend, 472, 474 
cause of tuberculosis of beet, 50 
Bacterium campestre (Pam.) EFS. (See 
also Black rot of crucifers), 145 
colonies, figures illustrating, 150, 

151 
cultural characteristics, 147, 153 
drying resistance to, 36, 147 
growth on agar, 147 
flagella of, 147, 150, 152 
host plants, 146 
in Holland and Denmark, 62 
inoculation by insects, 148 
inoculation experiments, 149 
isolation of, from diseased plant, 
149 



Bacterium campestre, literature, 159 
method of infection, 16 
method of inoculation, 150 
morphology, 147 
non-infectious to beans, 287 
resistant varieties of plants, 149 
retention of virulence, 148 
seed-borne, 36, 159 
type of infection, 16, 145 
Bacterium citri (Hasse) Jehle, 59, 60, 

61, 62, 66, 287, 329, 474 
Bacterium coronafaciens Elliott, cause 

of halo blight in oats, 474 
Bacterium delphinii, factors favoring 

infection, 13 
Bacterium glycineum Coerper, cause of 

blight of soy bean, 474 
Bacterium hyacinthi Wakker, 17, 62, 

473 
Bacterium juglandis (Pierce) EFS, 55, 

329, 473 
Bacterium lachrymans Smith and 
Bryan, 474 
cause of angular leaf-spot of cu- 
cumber, 36 
factors favoring infection, 13 
figures illustrating, 14, 15, 37, 38, 

39, 40 
prevalence of, 69 
Bacterium leguminosarum (Frank)EFS, 
action of, on plant tissue, 
46 
a polar flagellate organism, 46 
marked viscidity of cultures, 139 
Bacterium maculicolum McCulloch. 
(See also Cauliflower, spot of), 
300 
aerobe, 306 
appearance of cauliflower infected 

with, 300 
attacks cabbage also, 300 
behavior in various media, 306-310 
cause of cauliflower spot, 304 
colonies of, 303, 309 
dry air sensitive, 310, 316 
flagella of, 313 
green fluorescence, 306 
heat sensitive, 310, 316 
inoculation experiments, 31] 
lab ferment, 306 



668 



INDEX 



Bacterium maculicolum, liquefj'ing, 

308 
literature, 313 
method of obtaining pure cultures 

of, 310 
motility, 304 

sodium chloride sensitive, 306 
strata in milk and litmus milk, 308 
sunlight sensitive, 310, 316 
technical description of, 304-310 
temperature relations, 308 
type of disease caused by, 300 
ty rosin, 306 
viiulence soon lost, 310 
Bacterium malvacearum EFS. (See 

also Angular leaf-spot of cotton), 
314 
beans, non-infectious to, 329 
cabbages, non-infectious to, 329 
citius, non-infectious to, 329 
colonies, 329, 330, 332, 334 
comparison with other yellow 

organisms, 329 
contrast with Bact. phaseoli, 322, 

325 
description of, 321, 322, 324, 325, 

326. 327 
flagella, 325 
gardener's hose spreads organism, 

332 
gelatine plate colonies, 324, 335 
geographical distribution, 317, 319 
growth on oi in 

agar plates, 322 

beef peptone bouillon, 327 

blood serum, 325, 336 

litmus milk, 327 

milk, 327, 337 

potato, 326 

Uschinsky's solution, 327 
historj' of disease caused by, 314 
infections due to wind driven rain, 

322, 337 
inoculation experiments, 331 
lab ferment, 322 
literature, 339 
maximum temperature for growth, 

327 
means of distinguishing, 331 
method of infection. 16, 337 



Bacterium malvacearum, method of 

making pure cultures, 330 
non-growth at low temperatures, 

327 
resistant in tissues, 335 
sensitive to drying, 36 
sensitive to freezing, 328 
sensitive to sunlight, 36, 326, 327 
stomatal infections, 317 
thermal death point, 327 
type of infection, 317 
tyrosin, 327, 338 
windowed colonies, 324 
yellow saprophytes with, 330 
zoned colonies, 324 
Bacterium marginale Nellie A. Brown, 

cause of lettuce spot, 36 
Bacterium medacaginis (Sackett) EFS, 

474 
Bacterium mori Boyer and Lambert 

emend. EFS. (Cause of mul- 
berry blight), 340 
acid sensitive, 347 
appearance of plants infected with, 

340 
chloroform tolerant, 348 
cirri of, 340 
colonies, 356, 357 
description of, 347-351 
flagella, 353, 354, 355 
geographical distribution, 341 
growth on or in 

agar streaks, 348 

beef bouillon, 348 

blood serum, 348 

Cohn's solution, 348 

litmus milk, 348 

milk, clearing of, 348 

peptone water, 349 

potato, 348 

Uschinsky's solution, fluores- 
cent, 348 
history of disease, 342-347 
inoculation experiments, 351 
involution forms, 349 
loss of virulence in media slow, 351 
literature, 358 
method of isolation of pure cultures, 

351 
nomenclature, 342-347 



INDEX 



669 



Bacterium mori, non-liquefying, 348 
non-nitrate reducing, 347 
pseudozoogloese formed, 349 
thermal death point, 349 
sensitive to sunlight, 349 
sodium chlorid tolerant. 349 
tyloses due to, 477 
yellow saprophytes with, 342 
Bacterium phaseoli EFS. (See also 

Bean blight.) 
appearance of tissues inoculated 

with, 282 
bean varieties resistant to, 289, 

290, 291, 292 
behavior in milk cultures, 293 
cause of bean blight. 280. 285 
colonies of, 296, 298 
contrasted with Bact. malvacea- 

rum, 285, 287 
description of, 285 287 
distortions due to, 282, 284 
factors favoring infection, 13. 289 
flagella, 285, 294 
freezing, effect of, 293 
geographical distribution, 284 
growth in bean plant, 282, 284 
growth in various media, 285, 287 
inoculation experiments, 288 
lab ferment, 285 
liquefying, 285 
literature, 299 
method of obtaining pure cultures. 

287 
non-infectious to cabbage, 287 
non-infectious to citrus, 287 
resemblance to other yellow species 

of Bacterium, 287 
resists drying, 285 
starch destroyed by, 292 
sunlight, effect of, 295 
temperature relations, 285, 287, 

294 
treatment of plants inoculated 

with, 290 
virulence persists, 287 
winters over on seeds, 296, 297 
zoned colonies, 285, 324 
Bacterium oleae Archangeli, 343, 394 
Bacterium pisi (Sackett) EFS, 474 
Bacterium pruni EFS, 287, 329 



Bacterium pruni, cause of black spot 

and canker of plum and peach, 

13, 473 
method of infection, 16 
most susceptible age of host, 8, 10 
Bacterium savastanoi EFS. (See also 

Olive tubercle) 
acids formed. 400, 401 
aerobic, 400 
appearance of infected olive trees, 

389 
appearance of tissues infected with, 

391 
chloroform tolerant, 397 
colonies of, 402, 403, 405, 407, 

408 
cultural characters of, 396 
dwarfing of infected olive shoots, 

49, 392 
flagella, 396, 401 
formation of crystals in Cohn's 

solution, 407 
growth on or in 

agar plates, 397 

beef bouillon, 401 

Cohn's solution, 403 

Dunham's solution, 403 

gelatin plates, 398 

litmus milk (blued), 398 

milk cleared, 398 

potato, 398 

T'schinsky's solution, 401 

Winogradsky's solution, 403 
history of nomenclature, 394 
inoculation experiments, 404 
literature, 411 
loss of virulence on media not 

rapid, 404 
niaximum temperature for growth, 

399 
Merck's peptone harmful to, 401 
method of infection, 15, 389 
method of isolating from diseased 

tissues, 404 
methods of staining, 399 
non-hquefying, 396 
non-nitrate reducing, 396 
plants susceptible to, 394 
thermal death point of, 400 
transmission of, 21, 410 



670 



INDEX 



Bacterium sav'astanoi, undulate-erose 

colonies on gelatin, 398, 403, 405 

varieties of olive resistant to, 410 

Bacterium solanacearum EFS, 177. 

See also Brown rot of Solanaceae. 
aerobism, 186 

appearance of the disease, 178 
bipolar staining, 186 
colonies, figures illustrating, 190, 

191 
cultuial characteristics, 182 
dark stain on steamed potato, 186 
distortions of infected planes, 49, 

179 
duration of virulence, 34 
dwarfing of infected plants, 48, 49, 

184, 189 
flagella of, 192 
fluid colonies, 191 
geographical distribution, 182 
growth on or in 

agar, 186 

gelatin, 186 

non-growth in Cohn's solution, 
186 
host plants, 177 
inoculation technic of, 188 
insects may distribute, 199 
isolation of, from diseased plants, 

188 
literature, 200 

loss of virulence on media, 186 
loss of virulence in soils, 195 
method of infection in Sumatra, 21 
morphology, 182 
most susceptible age of host, 8 
motilit}', how best observed, 197 
nematodes and, 181 
on beans, 178 
on egg plant, 177 
on potato, 177, 178/^180 
on Ricinus, 184 
on sunflower, 185 
on tobacco, 180, 181 
on tomato, 178 
opalescent colonies of, 182 
plants for inoculation experiments, 

189 
pure cultures, method for obtain- 
ing, 188 



Bacterium solanacearum, recovery of 
plant from infection with, 18 
reduces nitrates, 186 
renders milk alkaline, 186 
roots, entrance through broken, 

181 
rotation of crops advised, 200 
sensitive to drying, 36, 186 
soil organism harmful to, 195 
stain in tissues due to, 180, 183 
stock cultures, care of, 188 
tyloses due to, 477 
two strains of, 188 
type of disease caused by, 177 
virulence lost quickly, 186 
weeds subject to, 178, 200 
well-water infected by, 21 
var. asiaticum EFS, 188 
Bacterium syringae (Van Hall) Giis- 
sow, 35, 473 
cause of lilac blight, 35 
Bacterium translucens Jones, Johnson 

and Reddy, 36, 329, 473 
Bacterium translucens var. undulosum 
Smith, Jones and Reddy, 474 
colonies of, 20, 26, 27, 28, 29, 31, 

32 
destroyed by formalin, 70 
effect on wheat kernels, 56 
resistance to drying, 36 
resistance to sunlight, 36 
signs of, on wheat heads, 22, 23, 

24 
variable severity of, 51 
Bacterium tumefaciens Smith and 
Townsend, 413. (See also Crown 

gall) 

acid-forming, 426 

action on plant tissue (hyper- 
plasial), 46 

animal inoculations with, 36 

appearance of tissues infected with, 
413 

cause of a disease in man, not es- 
tablished, 36 

chemical products of metabolism 
of, 457, 483, 558 

colonies of, 438, 461, 462 

Congo red absorbed by, 426 

cultural characters, 421 



INDEX 



671 



Bacterium tumefaciens, flagella, 459 
geographical distribution of, 413 
growth on or in 
agar, 426 
bouillon, filaments and stringing 

threads in, 426 
Cohn's solution. 426 
gelatin plates, 426 
litmus milk (blued), 426 
milk, casein slowlv separated, 

426 
potato, 426 

Uschinsky's solution, 426 
host plants, 430 

how distinguished from sapro- 
phytes in media, 438 
inoculation experiments, 438, 449 
involution forms, 428, 460 
isolation from diseased tissues, 

432, 433, 438 
lab forming, 426 
literature, 471 
may live in soils, 35 
mitochondria (?) confused with, 

419 
non-liquefying, 421 
non-nitrate reducing, 421 
non-starch destroying, 421 
occurs in tumor in small numbers, 

433 
plate cultures directly from tumor 

often develop slowly, 433 
saprophytes accompanying, 432 
sensitive to acids and alkalies, 426 
sensitive to germicides, 428 
serological reactions, 457 
several strains of, 457 
staining of, 466, 468 
temperature relations, 426 
transmitted by nursery stock, 469 
type of tumors produced by, 413 
virulence lost slowly, 428, 469 
virulence of colonies from tumors 
variable, 468 
Bacterium vascularum Cobb, 473 

dwarfing and distortion of sugar 

cane infected with, 49 
how avoided in New South Wales, 73 
Bacterium viridilividum Nellie A. 
Brown, cause of lettuce spot, 36 



Bacterium woodsii EFS, 16, 473 

method of infection, 16 
Balances, 78 

Banana, bacterial disease of, 473 
figure illustrating, 11 
Panama disease, 52 
rot of, in Philippines, 54 
in Sandwich Islands, 64 
Bark tumors in rubber trees (Hevea), 

478 
Barley, bacterial blight of, 55, 473 
Basal glume rot of wheat, 55. (See 

Bacterium atrofaciens.) 
Basal stem rot and tuber rot of potato, 
253. (See Bacillus phytophthorus 
and black rot of potato.) 
Basket willow, bacterial disease of, 53, 

474 
Bean, bacterial spot of. (See Bean, 

blight of.) 
Bean-blight, 280. (See also Bacterium 
phaseoli.) 
appearance of disease, 282 
bacterial crusts and cirri in, 282 
care of inoculated plants, 290 
causal organism oozing from tissues, 

282 
chiefl.y parenchymatic, 280 
chlorophyll may persist around leaf 

spots, 282, 283 
color changes in diseased plants, 282 
control of, 299 

description of causal organism, 285 
discolorations in, 282 
distortions due to, 282, 283 
dwarfings due to, 283 
etiology, 285 

figures illustrating, 280-288 
first signs of the disease, 282 
geographical distribution, 54, 55, 284, 

285 
Halsted's observation, 296 
histology, figures illustrating, 289, 

290, 291, 292 
host plants, 280 
inoculation experiments, 288 
isolation of causal organism, 287 
late stages of the disease, 282, 283 
literature, 299 
pathogen seed-borne, 297 



672 



INDEX 



Bean-blight, resemblance to other 
yellow organisms, 287 
rust-red margins of spots, 288 
seeds infected, 284, 297 
selection of material for sections, 294 
stomatal infection, 284, 285, 289 
stomatal ooze in, 282 
susceptible varieties of beans, 290, 

291 
temperature relations, 291 
time required to infect, 290 
troublesome nature of, 55, 285 
type of disease, 280 
Bees, agents in transmission of plant 

diseases, 25, 30 
Beetles, agents in transmission of plant 

diseases, 30 
Begonia, bacterial leaf spot of, 474 
Begonia hybrids and phyllomania, 575 
Begonia phyllomaniaca, 568, 571-628 
a hybrid. 575 

amount of water needed by, 615 
buds from trichomes, 616, 617 
comparison with B r y o p h y 1 1 u m 

calycinum, 619 
dwarfing of proliferous leaves, 594 
embryonic tissue red, 587 
experiments with, 574-631 
formation of adventive shoots, 575 
nearly free from, when un- 
disturbed, 599 
history of, 574, 575 
leaf distortions in, 620 
lenticels in, 615 
literature on, 629 
origin of plants experimented with, 

587 
phyllomania in, cause of, 575, 593 
figures illustrating, 586, 588, 591, 
594, 602 
proliferations at stipule scars, 599 
scarcity of stomata in, 615, 619 
sensitive to shock, 625 
shoots from internodes, 577, 582, 

589, 592, 598, 608 
shoots from leaf blades, 578, 584, 

604, 605, 611 
shoots from petioles, 596, 598, 606, 

610 
shoots from wounds, 613, 614 



Begonia phyllomaniaca, sub-epidermal 
storage system, 615 
watery nature of, 587 
Bibliographies, preparation of, 648 
Biochemistry, subsidiary studj' in 

pathology, 633 
Black arm, 314. (See Bacterium mal- 

vacearum.) 
Black chaff of wheat. (See also Bac- 
terium translucens var. un- 
dulosum.) 
distributed on seed, 20, 56 
introduced (?) from Russia, 66 
germicidal treatment of, 69-71 
prevalent in Central United 
States, 55 
Black-leg of potato, 253. (See Black 

rot of potato.) 
Black rot of crucifers, 145. (See also 
Bacteri u m campestre. ) 
brown or black veination of 

leaves, 146. 148 
cause of, 147 

chlorophyll, increase of, in, 157 
destructive nature of, 148 
dissemination by insects, 148 
dissemination by refuse from 

cabbage houses, 156 
dissemination by seed, 148 
dissemination from a seed bed, 

159 
etiology of, 147 
figures illustrating, 145, 146, 147, 

148, 149 
geographical di.stribution, 147 
histology, figures illustrating, 

154, 155, 156, 158 
host plants, 145 
introduced into United States 

from North Europe, 159 
literature, 159 
means of prevention, 159 
resistant varieties, 149 
slugs as carriers of, 159 
stain of vascular bundles in, 

146 
type of infection, 145 
variability of, 157 
water-pore infections common 
in, 146, 147, 148 



INDEX 



673 



Black rot of potato, 253. (See also 
Bacillus ph ytophthorus. ) 
appearance, 255 
base of stem specially subject to, 

254, 255 
black stain conspicuous in, 254, 

260 
cause of, 257 
contrasted with Bacterium so- 

lanacearum, 257 
control of, 278 

figures illustrating, 253, 254, 255, 
256, 258, 259, 260, 261, 262 
264, 275 
geographical distribution, 257 
histology, figures illustrating, 

263, 266 
host plants, 257 
inoculation experiments, 267 
leaf-curl in, 263 
lenticel infection, 265 
literature, 278 
method of isolation, 263 
non-starch consuming, 264, 266 
non-vascular disease, 253, 255 
resemblance to other potato dis- 
eases, 265 
resistant varieties, 277 
stomatal infections doubtful, 

277 
temperature relations, 274, 278 
transmisr-'on, 278 
type of disease, 253 
varieties subject to, 277 
virulence long persistent in or- 
ganism causing, 263 
weather relations, 257 
Blood serum, use as culture medium, 

105 
Blossom-blight, 16, 35&-361 
Blue prints, bleaching of, 128 
Boll-rot of cotton, 314. (See Bac- 
terium malvacearum.) 
Bordeaux mixture, 74, 114 
Braun (Harry), method of control of 

seed-borne organisms, 71, 72 
Brevity, advantage of, in scientific 
papers, 647 
when not desirable, 647 
Bromide prints, bleaching baths for, 128 
43 



Brooks (Win. K.), citation from, 646 
Broom corn, leaf-stripe of. (See Bac- 
terium andropogoni), 473 
Brown rot of Solanaceae, 177. (See also 
Bacterium solanacearum.) 
distribution of, 182 
figures illustrating, 177, 178, 179, 
180, 181, 182, 184, 185,' 186, 
187, 189, 276 
histology, figures illustrating, 

193, 194, 196, 199, 200 
in East Indies, 54 
in United States, 55 
widespread on many hosts, 177 
Brusone, rice disease common in Italy, 

66 
Bryophyllum, what sets leaf-buds 

growing, 619 
Bud-rot of coconut, 9, 52, 54, 474 
Buds, on tomato leaves, 50 

on crown galls, 419, 437, 439, 442, 
452, 456, 
Burrill (T. J.), discovers cause of pear 
blight, 1 

Cabbage, black rot of, 145. (See 
Black rot of cruciferous 
plants.) 
in Holland, Denmark and United 
States, 62 
tumors due to freezing, 485 
tumors due to sand-blast, 489 
Cabbage pith, woody cylinder in, 485 
Cabbage spot disease, 300. (See cauli- 
flower spot.) 
Calla lily rot, resemblance to soft rot of 

carrot, 229, 230 
Cameras, for general purposes, 90, 92, 
93 
Crandall model, 91 
for use in making photomicrographs, 
94 
Canada, bacterial diseases found in, 54 
Cancer 
in plants, 413. (See Crown, gall.) 
literature cited, 573 
of rats, 571 

parasite (?) suspected cause of, 570 
plant tumors suggestive of, 51, 417, 
419, 421, 471, 558, 569 



674 



INDEX 



Cancer, pre-cancerous stage, 483, 511, 
571 
theory as to cause of ,'569 
Cane diseases 

Australian, 52 
East Indian, 52 
Fiji, 52 

Porto Rican, 476 
South American, 54 
Canker of peach and plum, 13, 473 
Cankers on plants as a result of bac- 
terial infection, 50, 202 
Capsule spot of cotton, 314. (See Bac- 
terium malvacearum.) 
Carbol fuchsin, 117 
Card catalogues, vise of, 130 
Carnations, leaf spot of, 473. (See 
Bacterium woodsii.) 
new type of infection found in, 48 
Carnoy, fixative, formula of, 115 
Carrot, soft rot of, 223. (See Bacillus 

carotovorus.) 
Catalogues, 139 
Cauliflower, 

black rot of, 145. (See black rot of 

crucifers.) 
Harrison's soft rot of, 239 
McCuUoch's spot of, 300. (See also 
Bacterium maculicolum.) 
appearance of diseased tissues, 

300 
cause of disease, 304 
cool weather disease, 308 
description of pathogen, 304, 

306, 308 
figures illustrating, 301, 302, 307 
geographical distribution, 304 
histology, figure illustrating, 305 
history of the disease, 304 
immunity of very young and 

very old leaves, 302 
incubation period short, 302 
inoculation experiments, 311 
literature, 313 

method of isolating causal or- 
ganism, 310 
period of incubation, 302 
stomatal infections in, 300, 301 
type of infection, 300 
tumors due to chemicals, 551 



Cauliflower, tumors formed by sand- 
papering, 489 
Cavara (Fridiano), 2 
Cell-division, caused by lack of oxygen, 

51^ 
Cell-paralysis and cell-stimulus in tu- 
mor formation, 566 
Celery, blight of, 474 

rot, common in England and United 
States, 64 
figures illustrating, 67, 244 
losses from, 51 
soft rot of, 240 
Cellulose, doubtful action of pathogenic 

bacteria on, 46 
Centrifuges, use in Pathological Labora- 
tory, 78 
Chemical cell-stimulation, in chestnut 

wood, 478 
Chemicals, necessity for purity of, 107 
Chemicals produced by crown gall, 483 
Chemical stimulus resulting in tumor 

formation, 510 
Chemistry, need of, in pathology, 634 
Cherry, Barss' blight of, in Washington 

and Oregon, 474 
Cherry, German blight of, 474 
Chestnut, tyloses in wood of, 478 
China, bacterial diseases in, 53 
Citrus canker, 61, 474 

appropriations by Congress to con- 
trol, 61 
distribution of, 52, 62 
figures illustrating, 59, 60, 61, 62 
in Australia, 52 
in Japan, 53 
in India, 54 
in Phillippines, 54 
inspections in Florida for, 62 
Cladosporium citri Massee, 62, 65 
Clean hands, importance of, 108 
Clear ideas, 644 
Clearness, necessity of, in scientific 

writing. 643 
Coconut, bud-rot of, 474 
figure illustrating, 9 
in Philippines, 54 
in South America, 54 
in West Indies, 52 
Coleus, freezing experiments with, 566 



INDEX 



675 



Colletotrichum gossj'pii Southworth, 

321 
Colonies, planar enlargements with 

oblique light, 111 
Color changes in plants, due to bacterial 

infection, 49 
Color-chart used, 132 
Competing saprophytes, 74 
Confucius, sayings of, 637, 653, 656 
Control, methods of, 68 
Cooperation, a necessity in modern re- 
search, 656 
Copper sulphate treatments, 69 
Cork-formation, as a result of bacterial 

infection, 50, 233 
Corrosive sublimate treatments, 69, 

159, 176, 386 
Cotton, angular leaf-spot of, 314. (See 
also Bacterium malvacearum.) 
black arm and boll rot, other 

forms of, 314 
description of causal organism, 

321 
distortion of leaves due to, 282 
early stages of the disease, 316 
economical importance, 321 
figures illustrating, 314, 315, 316, 
318, 319, 321, 322, 323, 324, 
333 
geographical distribution, 54, 55, 

317, 319 
histologj', figures illustrating, 

320, 338 
history of disease, 314 
infection may be early, 316 
inoculation experiments, 331 
late stages of the disease, 317 
literature, 339 

method of isolating the or- 
ganism, 330 
pnethod of transmission, 337 
prevalent in China, 319 
prevalent in South Africa, 54, 

317, 319 
prevalent in Turkestan, 317 
spot disease versus vein disease, 

316, 317 
stomatal infections in, 317 
type of disease, 314 
Crandall camera for field work, 91 



Crown gall, 413. (See also Bacterium 

tumefaciens.) 
a hyperplasia, 413 
animal inoculations with organism 

causing, 36 
bearing leafy shoots, 419 
cells not killed by the parasite, 413 
chlorophyll in tumor strand, 459 
conversion of adjacent normal cells 

into tumor cells, 431 
crushing effect of tumor cells, 468 
description of causal organism, 421 
disorientation of cells in, 455, 460, 

467, 470 
dwarfing of infected plants, 49 

414, 415, 417, 436 
early stages, 459 
effect on host plant, 417 
embryomas, illustrations of, 435, 

436, 437, 439, 440, 442, 443, 444, 

447, 448, 452, 456, 458 
etiology, 421 
figures illustrating, 413, 414, 415, 

416, 418, 420, 422, 423, 432, 

450 
flower buds from, 437 
found in France and Italy, 66 
found in South Africa, 54 
geographical distribution, 413, 421 
growth, extra physiological, 417 
growth from cambium, 417 
growth from cortical parenchyma, 

417 
growth stimulus extending be- 
yond the tumor cells, 460, 468 
hairy root, 421 
histology, figures illustrating, 428, 

429, 430, 431, 433, 434, 443, 454, 

455, 464, 465, 467, 470 
host plants, 430 
inoculation experiments, 438, 449, 

451 
invasive tumor cells, 460, 468 
killing effects, 449 
lignin on bark parenchyma cells, 

430 
literature, 471 

method of control in rose houses, 73 
method of infection in South 

Africa, 15 



676 



INDEX 



Crown gall, method of isolating organ- 
ism, 432-438 

non-infectious colonies from, 468 

nurserymen common distributors 
of, 469 

olive resistant to, 469 

on chestnut, 413 

onion resistant to, 469 

on oak (undetermined), 414 

on rasin grapes in California, 417 

on wild fig, enormous size of, 414 

on willow in Africa, 417 

parasite intracellular. 419 

parasite, how carried in the tissues, 
419 

parasite, occurs in small numbers, 
433 

Peklo's studies, 463 

peroxidases in, 457, 563 

phenomena suggesting cancer, 51, 
558, 569 

prolonged incubation period in an 
orange tree, 17, 448 

respiration accelerated in, 563 

roots from, 421, 458 

secondary etiology of, 558 

secondary tumors from primary 
tumor with structure of latter, 
417 

secondary tumors, time required 
for development, 459 

shoots from, 419, 439, 440, 442, 
444 

stages in development of, 563 

staining diseased tissues, 466 

starch stored in, 563 

stem-structure of secondary tu- 
mors in leaves, 428, 429 

strains (several) of organism caus- 
ing, 457 

sugar abundant in, 563 

tracheids in, from stem-, leaf- and 
fruit-parenchyma, 431, 455, 463 

transmission of, 469 

tumor strand, figures illustrating, 
418, 424, 425, 427 

tumor strands in sunflower, 449 

type of disease, 413 

when excessively vascular, 468 

where found on the plant, 421 



Crown gall, wound disease. 469 

young tissues produce largest tu- 
mors, 417, 449 
Cruciferous plants, black rot of, 145, 

(See Black rot of crucifers.) 
Cryptogams, bacterial diseases of, 4 
Cucumber, angular leaf spot of. (See 
Bacterium lachrymans Smith and 
Bryan.) 
Cucumber wilt, 132. (See also Bacillus 
tracheiphilus.) 

carried by striped beetles, 30 

etiology, 132 

geographical distribution, 132 

literature, 145 

type of disease, 132 

winter occurrence of, 132 
Cucurbit wilt 

controlled by destruction of insects, 
74 

figures illustrating, 133, 134, 135 

histology of, figures illustrating, 
141, 142, 143 
Cultures of bacteria, care of, 80, 109 

isolation of, 80 

staining of, 118 

study of, 110 

tools for isolation of, 80 

transfer chambers for making, 80 
Culture media, 

agar, 105 

blood serum, 105 

bouillon, 105 

gelatin, 105 

milk, 104 

peptone water, 106 

preparation of, 77, 100 

pure sugars for, 107 

raw vegetables, 103 

steamed vegetables, 101 

synthetic, 106 

uses of, 99 

vegetable juices, 103 
Cuttings, disease transmitted by, 73 
Cyanide of mercury for pear blight, 71 

Dacus oleae (Rossi) Meigen, 407, 411 
Dark room, for photography, 95 
Denmark, bacterial diseases found in, 
62 



INDEX 



677 



Developer, 

for ordinary photographs, 95, 122 
for use in making photomicrographs, 
95, 125 

Diabrotica vittata Fabr., agent in trans- 
mission of cucumber wih, 30 
Bacilhis tracheiphihis winters over 
in, 33 

Distortions, as a result of bacterial in- 
fection, 49 

Drainage, importance of, 74 

Drawings, essentials of good, 129 
preparation of, 126-129 

Dry plates recommended, 95, 121 

Dubois (R), experiments in chloro- 
forming plants, 624 

Duplication of work, no danger of, 641 

Dutch East Indies, bacterial diseases 
found in, 52 

Dwarfing, as a result of bacterial infec- 
tion, 48, 49 

Effect of cold, heat, anesthetics, litera- 
ture, 630 
Emerson, wisdom of, 653 
Entomology, subsidiary study in plant 

pathology, 634 
Eel worm, galls due to, 543 
Enviromnent, effect of changes of, on 

parasite, 12 
Enzj-mes, excess of in diseased potato 

shoots, 543 
Erythrina, root disease of, 52 
Ether, as a stimulus to dormant buds, 

609, 622 
Ethics of research, 648 
Experimental method, importance of, 

638 
Experiments necessity for repetition of, 

637, 638, 640, 642 
Eyepieces, Zeiss, 85 
Eyestrain, 661 

Field corn, attacked by Stewart's dis- 
ease, 160 

Filing systems, 131 

Filters 

Berkefeld, 79 
Chamberland, 79 



Fire, use of, in isolating bacteria from 

tissues, 107 
Fire-blight of apple, pear, quince, etc. 

(See also Bacillus amylovo- 

rus), 359 
a bark disease, 359, 365 
a blossom-blight, 359 
appearance of diseased tis- 
sues, 359 
bacterial ooze in, 365 
Burrill's studies of, 1, 367 
control of, 365, 367, 385 
description of causal organism, 

367-372 
disintegrating action, 365 
early stages of disease, 359 
economic importance of, 55, 

365 
etiology, 367 

figures illustrating, 360, 361, 
362, 363, 364, 366, 367, 368, 

369, 370, 375, 383 
following crown gall, 384 
geographical distribution, 365 
germicidal treatment, 71, 386 
hailstone infection, 385 
histology, figures illustrating, 

371, 372, 373 
history of the disease, 359, 360 
hold-over blight or winter 

stage, 360 
home in United States, 385 
host plants, 359 
inoculation experiments, 374 
insects transmit, 360, 384, 385 
isolation of causal organism, 

374 
late stages of the disease, 360 
literature, 387 
means of detecting hold-over 

bhght, 381 
nectarial disease, 360 
on wild shrubs in United 

States, 359 
orchard destroyed by, 375 
parenchymatic disease, 359 
pruning for prevention, right 
sort of, 367, 376 
wrong type of, 362, 367 
Reimer's studies of, 386 



678 



INDEX 



Fire-blight, resistant varieties of pears, 
377, 383, 386, 387 
Sackett's studies of, 363 
secondary infections, 363 
soils favoring development of, 

383 
susceptible varieties of plants, 

377 
transmission by aphids, 384 
transmission by bees, 384 
transmission by birds, 384 
transmission by pruning tools, 

384 
type of disease, 359 
varieties of pears immune to, 

386, 387 
Waite's studies of, 25, 359, 360, 

372 
waterpore infections, sus- 
pected, 384 
water sprouts favor, 384 
Fire-heated soil, 73 
Fish, tumors in, 36 
Fixative, Carnoy, 115 
Flagella, staining of, 118 
Flemming's triple stain, 117 
Flowers, infection through, 16 
Forcing plants, by ether, by cold, and 

by warm bath method, 622, 623 
Formaldehyd, method of use for con- 
trol of plant diseases, 69 
Formalin treatments, 69, 71, 73 
Formic acid, tumors formed on plants 
by, 483 
figures illustrating, 498, 499, 500, 
501 
France, bacterial diseases in, 66 
Fraxinus excelsior, tumors on, 391, 394 
Freezing, tumors due to, 485 

figures illustrating, 504, 505 
Fundamental doctrine of science, 640 
Fungi followed by bacteria, 34 
Fungus infested soils, 114 
Fusarium cubense EFS, cause of the 

Panama banana disease, 52, 54 
Fusarium sp., following crown gall, 34 



Gall formation, theory of, 549 
Gas production, by bacteria, 36. 
also Bacteria in plants.) 



(See 



Gelatine, used for culture media, 105 
Generic names, how used in this book, 

1.32 
Germany, bacterial diseases in, 64 
Germicidal sprays, 74 
Germicides, use of, in isolating bacteria 

from tissues, 107 
Giant cells, 543, 545, 550, 565 
Glassware, in common use in plant 

pathology, 77 
Glomerella gossypii (Southw.) Edger- 

ton, 321 
Gram's stain and variants, 117 
Grand Rapids disease of tomato, 202. 

(See Aplanobacter michiganense.) 
Grape, crown gall on, 417 

intumescences on leaves of, 496 
Grass, western wheat-, disease of, 47, 

473 
Great Britain, bacterial diseases in, 64 
Gummosis of cotton, 314. (See Bac- 
terium malvacearum.) 

Hail stones, infection through wounds 

due to, 15, 385, 410 
Half-tones, preparation of, 122 
Hand lens, Zeiss, 86 
Herbarium specimens, care of, 119 
Heterodera radicicola Greef, cause of 

galls, 543 
Hevea brasiliensis Muell., brown bast 

disease of, 52, 476, 478 
Holland, bacterial diseases found in, 62 
Honing, on transmission of tobacco in- 
fection by well water, 21 
Hot air treatment of seed, 69 
Houser cabbage, disease resistant, 149 
Hutchinson (C. D.), disease of wheat in 

Punjab, 47 
Hyacinths, Wakker's disease of, 473 

yellow disease of, in Holland, 62 
Hyaloplasm, paralysis of, and tumor 

formation, 553 
Hybridization as a means for control 

of plant diseases, 75 
Hyperplasias in plants, chemicals a 
cause of, 483, 551, 563 
formed non-parasitically on potato, 

502 
freezing a cause, 485 



INDEX 



679 



Hj'perplasias in plants, gall flies a cause, 

552, 560 
increased acidity a cause, 515, 531, 

541 
myxomycetes a cause, 549 
nematodes a cause, 543 
primarily due to physical-chemical 

stimuii, 558, 563, 568 
result of bacterial infection, 51, 413 
semi-asphj'xiation a cause, 496, 

502, 511, 513. 519, 541 
wounding a cause, 489 
Hypertrophy, caused by physical- 
chemical stimulus, 508 

Ice boxes, use in culture work, 110 
Ice thermostat, 110 

Altmann's, 81 
Ideals of research workers, 651 
Illustrations, preparation of 
blue prints, 128 
drawings, 126-129 
engravings, 127 
paintings, 129 
photographs, 120 
photomicrographs, 123 
Imbibition, seed treatment by use of, 

preceding germicides, 71 
Immunity, acquired, 19 
Indexes, necessity for, 648 
India, bacterial disieases in, 54 
Incubation, period of, 16, 17 
Infection, 

birds, transmit, 25 
cages for, 111 

carried on seeds, 20, 202, 297, 337 
chemiotaxis and, 16 
distortion of host as a result of, 49 
due to wind-driven water, 21 
dung heap and, 21 
dwarfing of host as a result of, 49 
external factors governing, 13 
formation of overgrowths as a re- 
sult of, 50 
insects and, 25, 30, 74 
internal factors governing, 12 
rain or dew and, 21 
recovery from, 17-19 
spread by molluscs, 33 
spread bj' nematodes, 33 



Infection, through broken roots, 15, 16, 
181,212 
through hail storms, 15, 385, 410 
through natural openings, 16, 21, 146, 
160, 181, 202, 282-284, 300, 317, 
358, 360 
well water and, 21 
Inoculated plants, care of, 113 
Inoculation 

by use of insects, 112 
by soil, 111 
cages, 111 
methods of, 111 
syringes, 111 
time and place for, 112 
Insects, agents in transmission of plant 

diseases, 25, 30, 74, 159, 384 
Intumescences 
Atkinson on, 489 
due to acid stimuli, 508 
figures illustrating, 512, 514, 516, 517, 

518 
formed by lenticel obstruction, 496 
formed in absence of parasites, 477- 

510 
formed on potato in sealed tubes, 506 
Harvey on, 485 
literature, 572 
Sorauer's work on, 489, 491 
Tubeuf on, 631 
Viala and Pacottet on, [491 
Von Schrenk on, 483 
Wisniewski on, 496 
Wolf on, 489 
Iris, soft rot of, 239 
Islands of wood in bark, 478 

figures illustrating, 481, 482, 484 
Isolation of bacteria, technic of, 80, 107 
Italy, bacterial diseases in, 66 

Japan, bacterial diseases in, 53 
Johannsen: etherization of dormant 

buds, 622 
Jones' soft rot of carrot, etc., 223. (See 
Bacillus carotovorus.) 
disease corked out in potato, 

253 
disintegration of tissues, 226, 

229, 238 
dwarfing due to, 228 



680 



INDEX 



Jones' soft rot. of carrot, figures illus- 
trating, 224, 225, 227, 228, 
229, 230 
geographical distribution, 223 
histology, figures illustrating, 

226, 231, 232, 233 
host reaction, 230 
one species or several as cause 

of, 223, 229 
on many hosts, 223, 225 
prevention, 252 
swift action, 224, 229 
turgid tissues most susceptible, 

225, 227 
wound parasite, 223 
Journals for publication, 643 
Juvenal, wisdom of, 661 

Kale, black rot of , 145. (See Black rot 
of crucifers.) 

Knee-shaped bendings, 49 

Knot of olive, 389. (See Olive tu- 
bercle.) 

Kohlrabi, black rot of, 145. (See 
Black rot of crucifers.) 

L.\RKSPUR, blight of, 474 

Leaf -spots, 8, 74, 280, 300, 314 

Latin and Greek, need of in pathology, 

632 
Legumes, root-nodules of, 46, 139, 551 
Lenses, for photographs, 91 

planar, 92, 126 
Lenticels, tumors formed in, 513 

figures illustrating, 512, 514, 516, 
517, 518, 525 
Lettuce, blight of, 474 
Lettuce diseases, methods for control of, 

74 
Lichens, apple twigs smothered by, 365 
Light filters, for use in photography, 94 
Lilac, blight of, 62, 473. (See also Bac- 
terium syringae.) 
Lime-sulphur spray, 74 
Longfellow, citation from, 642 
Losses due to bacteria, 51, 54 

Maize, Stewart's disease of, 160. (See 

also Aplanobacter stewarti.) 
Maladie d'Oleran, bacterial nature of, 66 



Mango, bacterial disease, (See also 

Bacillus mangiferae.) 

figures illustrating, 39 

in South Africa, 54 

Mango leaf-, stem-, and fruit-spot, 473 

Manihot, disease of, found in South 

America, 54 
Manure-heap, keep diseased rubbish 

out of, 74 
Massachusetts potato disease, 219. 

(See also net- necrosis.) 
Mathematics, as a subsidiary subject 

in pathology, 634 
McCuUoch's cauliflower spot, 300. 

(See also Cauliflower.) 
Media for cultures 
agar, 105 
blood serum, 105 
gelatin, 105, 106 
milk, 104 

peptone water, 106 
preparation of culture media, 100 
Soyka's, 230 
steamed vegetables, 101 
synthetic, 106 
uses, 99 

vegetable juices, 103 
Medicine, knowledge of, useful to plant 

pathologists, 635 
Mercuric chloride treatments, 69, 159, 

176, 386 
Metabolism, products of bacterial, cause 

overgrowths, 483 
Methods of control, 68 
Methods of research, 76 
Meyer (Frank N.), discovers tobacco 
wilt in China, 53 
resistant pear trees collected by, 
377 
Micrococcus amylovorus Burrill, 367, 

388 
Microscopes, 
eyepieces for, 85 
objectives for, 85 
Zeiss, 84 
Microscopic preparations, how pre- 
served, 120 
Microtomes, 

Minot precision, 83 
Minot Rotary, 83 



INDEX 



681 



Microtomes, Reinhold-Giltay, 82 

Spencer rotary, 83 
Milton, citation from, 652 
Modern languages, need of, in patho- 
logy, 633, 650 
Modern science, value of, 642 
Molisch, Hans, forcing dormant buds 

with warm water, 623 
Molluscs, diseases transmitted by, 33 
Montaigne, wisdom of, 633, 652 
Mottling of leaves due to loss of water, 

506, 553 
Mulberry blight, 340. (See also Bac- 
terium mori). 
appearance of diseased plant, 340 
dead twigs due to, 341 
description of pathogen, 347 
distortion of leaves, 340, 342 
early stages of disease, 340 
figures illustrating, 341, 342, 343, 

344, 345, 346, 350 
geographical distribution, 341, 342 
histology, figures illustrating, 347, 

349, 352, 353 
history of disease, 343 
inoculation experiments, 351, ?54 
late stages of disease, 340 
literature, 358 

method of isolating causal or- 
ganism, 351 
resemblance to pear blight, 340 
susceptible varieties of mulberry, 

341 
tyloses in, 357, 477 
type of disease, 340 
Miiller-Thurgau, potato experiments, 

622 
Museum specimens, 120 
Mushrooms, bacterial rot of, 66 
Mustard, black rot of, 145. (See black 
rot of crucifers.) 



Nectaries, infection through, 16 
Negatives, 

filing of, 131 

labeling of, 131 
Nematode galls, 543, 550 
Nematodes, as agents in transmission 

of diseases, 33, 181 



Nematodes in soils, treatment of, 73, 
114 

Ngmec, studies of nematode tumors, 
545, 549 

Net-necrosis of potato, 218-221 

Nigrosin, use of, in staining, 117 

Nitrate reducing organisms, 147, 182, 
230, 257 

Non-nitrate reducing organisms, 135, 
161, 205, 285, 306, 347, 367, 396, 421 

Nucleus, division of, in tumor forma- 
tion, 545 

Nurserymen, responsible for disease, 19, 
20 



Oak, frondose gall on, 556, 557, 559, 

560, 561, 562, 564, 565 
Oats, halo-blight of, 55, 474 
Objectives, Zeiss, 85 
Observation and reflection, insufficiency 

of, 637 
Oedema of tomato, 489 

figures illustrating, 508, 509 
O'Gara (P. J.), disease of western wheat 
grass, 47, 473 

on fire blight, 365, 371, 381, 384, 386 
Oleander tubercle, 33, 394 
Olive knot. (See olive tubercle.) 
Olive tubercle, 389. (See also Bacte- 
rium savastanoi.) 
appearance of diseased tissues, 389 
bacterial ooze abundant, 389 
cavities formed in tissues, 389 
channel of secondary infection, 396 
etiology, 394 
figures illustrating, 390, 391, 392, 

393, 395 
geographical distribution, 66, 391 
German views, concerning, 395 
granulomatous nature of, 389 
histology, figures illustrating, 396, 

397, 398, 399 
history of the disease, 391 
in what fields and climates most 

abundant, 410 
literature, 411 

method for control of, 74, 410, 411 
method of isolating causal organ- 
ism, 404 



682 



INDEX 



Olive tubercle, nomenclature of organ- 
ism causing, 394 
no tumor strands in, 389 
structure of, 389 
type of disease, 389 
wild olives subject to, 389 
wound infection, 389 
Opium poppy, bacterial disease of, 54 
Orchard grass, new type of infection 
found in, 48 
Rathay's disease of, 473 
Osier (Sir Wm.), wisdom of, 661 
Oven, for sterilizing glassware, 78 
Overgrowths, as a result of bacterial 
infection, 50 
how otherwise produced, 477 
Oxygen and the bacterial cell, 515 
and the tumor-cell, 511, 531, 541 



Paintings, for illustration, 129 
Panama disease of banana, 52 
Panchromatic plates, use of, in photo- 

micrographic work, 95 
Paraffin oven, 83 

Parasites, sensitive to environment, 12 
action on tissues, 46 
dry air sensitive, 135, 195, 231, 

257, 306, 322, 372, 421 
dry air resistant, 147, 161, 207, 

285, 348 
effect of freezing on, 285, 306, 

322, 372 
effect of moisture, 13 
effect of shade, 13 
effect of sunlight, 156, 231, 285, 

306, 322, 349, 369, 404, 421 
effect of winds, 13 
extracellular versus intracellu- 
lar, 46 
killing temperatures, 135, 139, 
155, 214, 231, 272, 285, 308, 
327, 348, 371, 399, 426 
soon followed by saprophytes, 34 
summer temperatures, stimula- 
ting effect of, 13 
summer temperatures repressive 
effect of, 310 
Parasitism versus symbiosis, 41 
Pasteur, wisdom of, 642, 656 



Pavetta nodules, figures illustrating, 42 , 

43, 44, 45 
Pea, stem blight of, 474 
Peach, black spot of, 473. (See Bac- 
terium pruni.) 
Pear blight, 359. (See also fire-blight 
and Bacillus amylovorus.) 
discovery of bacterial origin of, 1 
economic importance of, 54 
enormous losses due to, in United 

States, 55 
how spread, 68 
in stone fruits also, 69, 359 
methods of control, 68, 385 
occurs now in Japan, 365 
Pelargonium, leaf-spot, 474 
Peptone water, use as culture media, 

106 
Peroxidase in tumors, 563 
Pethybridge and Murphy, potato rot of, 

64 
Phenolphthalein, how used in titrations, 

106, 132 
Philippines, bacterial diseases found in, 

54 
Photographs, preparation of, 120 
Photography, 
cameras, 90 
lenses, 91 
room for, 89, 96 
value of, 121 
washing devices, 95 
Photomicrographs, camera for use in 
making of, 94 
developer for, 125 
fixing bath, 125 

light filters, use of, in making, 94 
preparation of, 123 
time of exposure for, 124 
Phyllomania in Begonia, 574-630 
abnormalities observed in, 615 
causes distortions, 612 
comparison with effect of anaesthe- 
tics on dormant buds, 622 
comparison with effect of cold, 622 
comparison with effect of heat, 623 
due to shock, 575 
epidermal nature of, 583 
from glands and trichomes, 583 
from wounds, 590 



INDEX 



683 



Phyllomania in Begonia, not a winter 
state, 575 
proliferations not mirror images, 

612 
nutrition and, 581 
root-injury causes, 593 
stage of greatest susceptibility, 579 
theory concerning, 600-607, 609 
top pruning causes, 609 
Physics, a subsidiary study in patho- 
logy, 633 
Phytophthora infestans(Mont.)de Bary, 

bacteria follow, 34 
Pierce (N. B.), control of walnut blight, 

74 
Piricularia on rice, 66 
Planar lenses, 92, 126 
Plant cancer, 413. (See crown gall.) 
suggested relation of, to animal 
cancer, 569, 571 
Plant parasites, general morphology 

and cultural characters, 35 
Plant, physiology, importance of, to 

pathologists, 634 
Plasmodiophorabrassicae Woronin, 549, 

551 
Plates, lumiere, 126 

Plum, black spot of, 473. (See Bac- 
terium pruni.) 
Poppy, bacterial disease of, 54 
Portugal, bacterial diseases in, 66 
Potato, 

Appel's rot of, 253 

basal stem rot of, 253. (See also 

Bacillus phytophthorus.) 
black leg of, 253. (See black rot and 

Bacillus phytophthorus.) 
black rot of, 253 
brown rot of, 177 
intumescent shoots, 

excessive acidity of, 528, 536, 543 
excessive sugar content of, 541, 

563 
figures illustrating, 532, 533, 534, 
535, 536, 537, 538, 539, 540, 
542, 544, 546-548 
lenticel proliferation in, 502 
net-necrosis of, 219 
rots, 

found in Australia, 52 



Potato, rots found in England, 64 
found in France and Italy, 66 
found in Germany, 64 
found in United States and Canada, 

54 
general discussion of, 263-266 
great looses due to, 54, 64, 66 
Spieckermann's ring rot of, 207, 474 
Printing papers, 122 
Proliferation, in Begonia, 574-630 

abundant over mid-ribs, 596, 604, 

605, 606, 612 
arising from epidermal hairs, 583, 

616, 617 
arising from epidermis, 583, 618, 

621 
arising from internodes, 577 
a teratosis, 579 

caused by a definite shock, 575 
caused by loss of water, 593 
caused by wounding, 590, 593, 613, 

614 
changes in plant leading to, 600 
comparison with similar phenom- 
ena in other plants, 581, 619 
corking over of stimulated tissues, 

586, 589, 594, 607, 609, 616 
development of adventive shoots 
away from the mother plant, 
580, 581 
distortion resulting from, 612 
experimentally produced on inter- 
nodes, 582, 586, 589, 592, 598, 
608, 601 
extent of development of, 581 
from totipotent cells, 583 
general on leaf-blade, 578 
increase of acidity as a cause of, 

601 
literature on, 629 

nature of shock necessary to pro- 
duce, 599 
produced at will, 579 
season of year it may be pi'oduced, 

575 
shocked leaves not stunted, 584, 

594, 612 
shocked leaves stunted, 602, 611 
table of number of shoots on stimu- 
lated internodes, 627 



684 



INDEX 



Proliferation, in Begonia, table of results 
of wounding leaf blades, 626 

Pre-soak seed treatment, 71 

Prophylaxis, 68 

Protea, leaf spot of, 66 

Pseudomonas tritici Hutchinson, cause 
of wheat disease in India, 48 

Ptah-Hotep, precepts of, 653 

Publications, brevity desirable in, 646 
clearness essential, 644 
good summary essential, 647 
place of publication important, 643 

Pyrus ussuriensis Maxim., resistant to 
pear blight, 377, 386, 387, 388 

Rand (F. V.), experiments with bac- 
terial wilt of cucumbers, 30 

Rape, black rot of, 145. (See black rot 
of crucifers.) 

Rathay's disease of orchard grass, 62, 
473 

Razors, for microtome use, 83 

Recovery from disease, 17-19 

Red cabbage, freezing experiment with, 
566 

Reimer's resistant pear stocks, 386, 387 

Reimer's treatment of pear blight, 71 

Research, ethics of, 648 

Resistance, to disease, in plants, 16, 75 

Rice disease, nature of, in Italy, 66 

Ring rot of potato, 474 

Root-nodules, of legumes, 46, 551 

Roots, infection through, 15, 16, 188, 
212 
from crown gall, 421, 458 

Rose galls, how avoided, 73 

Rotation, as a method of control in 
plant diseases, 74 

Rubber, brown bast disease of, 52, 476 

Russia, bacterial diseases in, 66 

Saint Paul's advice, 658 
Salamanders, inoculations of, 36 
Sand-blast, hypertrophies due to, 489, 

507 
Sandwich Islands, bacterial diseases in, 

64 
Savastano (Luigi), 2, 400 
Scientific man's stock in trade, 654 
Sections, free-hand, 114 



Sections, fugitive stains, 117 

microtome, cutting and care of, 115 
fixing tissues for, 115 
keeping record of, 115 
staining of bacteria in, 116 
filing of, 120 
preparation of, 81, 114 
stains recommended for, 117 
straightener for, 83 
technic of making, 115 
Seed-beds and disease, 73, 159 
Seeds, treatment of, 69, 71 
Seedsmen, responsibility for spread of 

disease, 19, 20 
Seneca, wisdom of, 639 
Sereh of cane, 52, 476 
Sharing credits, 657 
Shriveled seeds and disease, 56, 69 
Similarity and identity, fallacy of, 640 
Soft rot of carrot, 223. (See also Bacil- 
lus carotovorus.) 
appearance of diseased tissues, 

224 
description of causal organism, 

230 
etiology possibly confused, 229, 

230, 232, 235 
figures illustrating, 224, 225, 227, 

228, 229, 230 
geographical distribution, 223 
histology, 226, 231, 232, 233 
inoculation experiments, 241 
literature, 252 

method of isolating causal or- 
ganism, 241 
other host plants, 225 
resemblance to calla lily rot, 229 
resemblance to other soft rots, 

223, 239 
type of disease, 223 
Soil, 

and infection, 21, 34 
method of sterilizing, 86 
Solanaceae, brown rot of, 177. (See 
also Bacterium solanacearuvi.) 
appearance of disease, 178 
cause of disease, 182 
dwarfing due to, 181, 184, 189 
figures illustrating, 177, 178, 179, 
180, 181, 182, 183, 185, 186, 187 



INDEX 



685 



Solanaceae, brown rot of, geographical 
distribution, 182 
histology, figures illustrating, 
193, 194, 196, 197, 198, 199. 
200 
history, 177 

host plants numerous, 177 
inoculation by needle pricks, 188 
literature, 200 
method of isolating pathogen, 

188 
plants and cultures for artificial 

inoculation, 189 
type of disease, 177 
Sorghum, leaf-stripe of, (See also Bac- 
terium andropogoni.) 
South Africa, bacterial diseases found 

in, 54 
South America, bacterial diseases found 

in, 54 
So}^ bean, leaf spot of, 474 
Spain, bacterial diseases in, 66 
Specimens, care of, 1 19 
Spieckermann's disease of potato, 64, 

207, 474 
Spraying for control of disease, 74 
Staining, 

bacteria, 116, 118 
flagella, 118 
jars for, 84 
methods, 116 
Stains, 

Carbol fuchsin, 117 
Flemming's triple, 117 
Gram's, 117 
Griibler's, 84 
methj'lviolet, 117 
nigrosin, 117 
special, 119 
Starch, 

removal by the plant from vicinity 

of the diseased tissues, 50, 233 
storage of, in diseased tissues, 50, 541, 
547, 563 
Stasis of circulation and tumors, 541 
Statesman and philosopher versus man 

of science, 636 
Steamers, used in sterilizing culture 

media, 78 
Steam-drag, 74 



Steam-heated soil, 73, 114 
Stereotyped thinking, 636 
Sterile cooperations, 657 
Sterilization 
by fire, 107 
by germicides, 107 
of leaf surfaces, 351 
importance of still air, 108 
importance of clean hands, 108 
Stewart's disease, 160. (See also 
Aplanobacter stewarti.) 
appearance of, on sweet corn, 160 
cause, 161 

cultural characteristics of patho- 
gen, 161 
dwarfing, 170 
etiology of, 161 

field corn also attacked by, 160 
figures illustrating, 160, 162, 163, 

164, 170, 171, 174 
first signs, 170 

geographical distribution, 161 
histology, figures illustrating, 165, 

166, 167, 168, 169, 174, 175 
host plants, 167 
how disseminated, 176 
inoculation experiments, 165 
literature, 176 
means of prevention, 176 
morphology of pathogen, 161 
seed-borne, 161 
type of disease, 160 
white male inflorescence, 162 
yellow ooze from cut stems, 163 
yellow pockets in tissues, 164, 171 
Still air, importance of, in making iso- 
lations, 108 
Stomata, infection through, 16. (See 
also Infection through natural 
openings.) 
test for condition of, 553 
wide open over intumescences, 
543, 548 
Streptococcus viridans Schottmtiller, 

570 
Stripe-disease of broom corn and sorg- 
hum, 473 
Style, how acquired, 644, 645 
Subjects, suitable for special study, 474, 
475, 476, 477 



686 



INDEX 



Success, how attained, 659 
Sugar beet, 

crown gall on, 414, 420, 422 

leaf spot, 474 

Metcalf's soft rot, 474 

tubercle of, 50, 472, 474 
Sugar cane, 

bacterial disease of, in South 
America, 54 

Cobb's disease of, 52, 473 

period of incubation in, 17 

Fiji disease, 476 

Sereh, 52, 476 

stripe disease (mosaic), 476 
Sugar in tumors, 563 
Summaries, importance of, 648 
Supernumerary organs, 50 
Susceptibility, period of greatest, 8 
Swedes, rot of, 66 

Sweet brier, frondose gall on, 552, 554 
Sweet corn, Stewart's disease of, 160. 

(See Stewart's disease.) 
Symbiosis, 41 
Syringes, 111 

Team work, necessary in research, 656 
Temperature, arrangements for con- 
trolling, for culture work, 81 
Teratosis, production of in Begonia, 

574-630 
Teratoma, on potato, 508, 540 
Text-books recommended for study, 76 
Thermo-regulators, Roux, 81, 109 
Thermostats, 109 

Rohrbeck, 81 
Tyloses in plants, 357, 477 

figures illustrating, 200, 478, 479, 
480 
Titration, apparatus for, 80 
Titration of media, how practiced in 

my laboratory, 132 
Tobacco, 

bacterial disease of, in South Africa, 
54 

brown rot of, 177. (See also Bac- 
terium solanacearum.) 

disease of, in Sumatra and Java, 
economic importance of, 52 

leaf-spots of, 54, 476 

wilt, in China, 53 



Tobacco wilt, in Cuba and Porto Rico, 
182 
in Japan, 53 

in Southern United States, 55 
Tomato, 

bacterial canker of, 202. (See also 
Aplanobacter michiganense.) 
appearance of the disease, 202 
cause of the disease, 205 
figures illustrating, 203, 204, 205, 

206, 210, 211, 212 
geographical distribution, 205 
histology, figures illustrating, 

207, 208, 209, 213, 214, 215 
infectious nature of, 202, 216 
isolation of causal organism, 207 
literature, 222 

parasitic organism probably 

seed-borne, 202 
resemblance to a disease of pota- 
toes in Germany, 207 
resemblance to brown rot, 202 
restricted growth of pathogen in 

certain media, 206, 207, 217 
signs of the disease, 202 
staining of tissues, 202 
stomatal infection common, 202 
technical description of patho- 
gen, 205 
tissues attacked, 202 
transmission of disease, 202 
type of disease, 202 
brown rot of, 177. (See Bacterium 

solanacearum.) 
oedema of, 489, 508, 509 
wilt, found in South Africa, 54 
Topics suitable for special study, 474, 

475, 476, 477 
Toxins, production of, by bacteria, 46 
Transfer chambers for making cultures, 

80 
Transmission of disease, agents, 19-34 
by aphides, 159, 384 
by bees, 25, 30, 360, 384 
by beetles, 30, 134, 135, 199 
by bugs, 384 
by birds, 25, 384 
by bulbs, 20 

by diseased refuse, 159, 278 
by dung-heap, 21 



INDEX 



687 



Transmissionof disease, by flies, 384, 411 
by gardener's hose, 219, 332 
by grafts, 20, 469 
by hail, 410 

b}^ lepidotera larvae, 159 
by molluscs, 25, 33 
by nematodes, 33, 181 
by nursery stock, 62, 385, 469 
by pruning tools, 73, 384 
by seeds, 20, 56, 58, 148, 159, 

161, 216, 297 
by seedsman, 20, 159, 174 
by snails, 33 
by soil, 21, 195, 469 
by tubers, 278 
by well-water, dew, or rain, 21, 

411 
methods of control, 68 
wind driven rain, 21, 337 
worms, 25, 410, 411, 469 
Trap crops, 74 

Tubercles and tumors in plants as a 
result of bacterial infection, 50, 389, 
413 
Tubeuf on intumescences, 631 
Tumors, etiology of, more important 
than structure, 510 
excessive movement of foodstuffs to 

vicinity of, 519, 541 
formed by bacteria, 50, 389, 413, 558 
structure of, 51, 424, 425, 427, 
428, 429, 430, 431, 454, 455, 
464, 465, 467, 470 
formed by chemicals, 483, 551 

structure of, 491, 492, 493, 494, 
495, 497, 501, 503 
formed by freezing, 485 

structure of, 505 
formed by increase of acidity, 513, 

531, 543 
formed by loss of water, 513 
formed by nematodes, 543 

structure of, 550 
formed by oxygen-hunger, 513, 531 
formed bj^ sand blast, 489 

structure of, 507 
formed by semi-asphyxiation, 496, 515 
formed by wounding, 489 
formed in absence of parasites, 477- 
509 



Tumors, formed in sealed tubes, 502 

figures illustrating, 520, 521, 
522, 523, 524, 526, 527, 528, 
529, 530, 540 
formed not b}' strong light, 496, 539 
formed on potato shoots in sealed 
tubes 
figures illustrating, 532, 
533, 534, 535, 536, 537, 
538, 539, 540 
formed on potato shoots in the open, 

542, 544, 546, 547, 548 
general discussion of origin of, 510- 

572 
giant cells in, 543, 545, 550, 565 
higher type of, 413, 558 
hyperplasia!, non-parasitic, on 

potato, 506 
in begonia, 502 
in fig, 502 

in leaves of grape, 491, 496 
in mulberry, 502 
literature, 572, 631 
many classes of, 510 
physical-chemical stimuli underlying, 

510-572 
secondary causes, 511, 566 
stasis in tissues originating, 513, 531, 

541 
theory of cause, 511, 555, 566, 568 
Turnips, black rot of, 145. (See black 

rot of crucifers.) 
Tyloses, origin of, 477 

United States, bacterial diseases found 
in, 54 

Vactjum pumps, 80 

Varieties resistant to disease, 75, 135, 
149, 295, 377, 383, 386, 387, 410, 
411 
sensitive to disease, 167, 277, 290, 
291, 377, 410 
Vascular diseases, 132, 145, 160, 177 
tissue invasion in, 10, 141, 166, 
196 
Velox printing paper, 122 
Vine diseases in France and Italy, 66 
Virulence, loss of, 19 
symbionts and, 477 



688 



INDEX 



Von Faber (F. C), on Pavetta nodules, 

45 
Von Schrenk (H.), on intumescences, 

483 

Waite (M. B.), discovers transmission 
of pear blight by bees, 25 
finds "hold over" blight, 360 
photograph, 2 

Wakker (J. H.), 2 

Walnut blight, 473 
in Chili, 55 
in New Zealand, 55 
in South Africa, 55 
in Tasmania, 55 
in United States, 55 

Water-color drawings, 129 

Water pores, infection through, 16, 21 

Water pores, figures illustrating, 146, 
147 

Water, varying requirements of para- 
sites for, 13 
varying requirements of plants for, 
113 



Wehmer (C), on potato rots, 264 
West Indies, bacterial diseases found 

in, 52 
Wheat, basal glume rot, 57, 58, 474 
black chaff of, 19, 20, 22, 23, 24, 26, 
27, 28, 29, 31, 32, 56, 70, 474 
White spot of alfalfa (physiological), 61 
Willow, 

disease of Japan, 53, 474 
disease of South Africa, 423 
Wilt disease 

of cucurbitaceae in United States, 

55, 132 
of potato and tomato in Africa, 54 
of tobacco in United States, 55 
Witch-broom, on pine, result of bac- 

tei'ial infection, 50 
Woods' disease of carnation, 473 
Wordsworth, citation from, 634 
Worms, cause of tumors or galls, 543 

Yellow disease of hyacinths, 62 

Zoology, subsidiary study in plant 
pathology, 634 



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